Sex- specific selection of sperm from transgenic animals

ABSTRACT

The present invention relates to methods and materials for pre-selecting the sex of mammalian offspring. In particular, the materials and methods described herein permit the enrichment of X- or Y-chromosome-bearing sperm in semen by introducing a transgene into a sex chromosome under control of regulatory sequences that provide for expression of the transgene in a haploid-specific manner.

The present application claims priority to U.S. Provisional PatentApplication No. 60/278,155, filed on Mar. 22, 2001, which is herebyincorporated by reference in its entirety, including all tables,figures, and claims.

FIELD OF THE INVENTION

The present invention relates to methods for pre-selecting the sex ofmammalian offspring. In particular, the materials and methods describedherein permit the enrichment of X or Y chromosome-bearing sperm in semenby expressing a transgene present on a sex chromosome in ahaploid-specific manner.

BACKGROUND OF THE INVENTION

Throughout history, humans have sought the ability to assert controlover the sex of offspring; both human and livestock. Homo sapiens'attempts to select sex of offspring prior to conception has beenwell-documented, as evidenced by historical descriptions of methods.Early techniques, circa 500 B.C., began with monoorchydectomy andprogressed through a variety of techniques which have come down to usvia folklore (such as placing an egg or scissors under the bed forconception of a girl, and placing a hammer under the bed and tying offthe left testicle to conceive a boy) (Fugger, 1999, Theriogenology52:1435-1440). A more scientific approach began in the last century andincluded utilizing a reported differential survival between X and Yspermatozoa dependent on the pH of the medium. (Shettles, 1970). Furthertechniques progressed to exploit differences in motility (Ericsson etal., 1973, Nature 246:241-24, Steeno et al., 75, Botchan et al., 1997)or cell density (e.g., centrifugation in a Percoll gradient, Lin et al.,1998, J. Assist. Reprod. and Genetics 15:565-569) to use indistinguishing X from Y sperm. Other techniques tried include size, headshape, surface properties, surface macromolecules, mass, and swimmingvelocity (see review by Windsor et al., 1993, Reprod. Fert. Dev.5:155-71). One group, Fabricant et al., (U.S. Pat. No. 4,722,887),utilized the differential expression of a sperm cell-surfacesulfoglycolipid to develop a method for separating X-chromosome-bearingand Y-chromosome-bearing sperm by polymeric phase separation.

A recent approach to the problem of sex pre-selection relates to methodsthat rely on the use of antibodies directed to sex-specific epitopes onsperm, or, alternatively, on fertilized embryos. For example, evidencefor a male-specific cell surface antigen was first obtained by Eichwaldand Silmser (1955, Transplant Bull 2:148) using the inbred mouse strainC57BL/6, but it remained for Hauscha (Transplant Bull, 1955, 2:154) tolater hypothesize the existence an antigen coded for by a Y-linked gene.This surface marker became known as H-Y (bistocompatibility locus on theY chromosome). Y-sperm-specific surface expression of the H-Y antigenhas been suggested to be a target epitope for sex pre-selection, andantibodies raised to the H-Y antigen were expected to allow the routinesorting of sperm using cell sorting or immunological adsorption of H-Yexpressing sperm (Peter et al., 1993, Theriogenology 40:1177-1185).Similarly, sex-specific antibodies were disclosed as allowing theselective ablation of sperm or embryos utilizing complement (U.S. Pat.No. 5,840,504). See also, U.S. Pat. No. 4,999,283; U.S. Pat. No.4,511,661; U.S. Pat. No. 4,191,749; U.S. Pat. No. 4,448,767; U.S. Pat.No. 4,680,258; and U.S. Pat. No. 5,840,504.

The locus of at least one of the genes responsible for H-Y expression ison the Y chromosome, and this antigen has been shown to becross-reactive among numerous speciess ranging from fish to man. It ispossible that the H-Y antigen may be the primary sex determinant and maycontrol testicular development in mammals. (Wattle, et al., 1975; Wattleand Ok, 1980); Ok, et al., “Application of Monoclonal Anti-H-Y Antibodyfor Human H-Y Typing,” Human Genetics, 57: 64-67 (1981). H-Y is a“minor” histocompatibility antigen, which is a separate genetic locusfrom the major histocompatibility complex (MHC). Minorhistocompatibility loci are mainly concerned with cellular immunity; fewif any products of these loci are efficient in raising antibodies.Nevertheless, a search for a serological counterpart to thetransplantation H-Y antigen appeared to have been successful when aserological “E-Y” method was reported by Goldberg and coworkers (1971,Nature 232: 478). Recent data indicates, however, that the serologicaldetectable “H-Y” antigen may not be the same as the histocompatibilityantigen. (Simpson et al., 1990, Arch. Androl. 24:235). The moleculeidentified by serological methods is now widely referred to asserologically detectable male antigen (SMA).

These immunological methods have not always lived up to expectationshowever (Bradley, 1989). For example, some authors found no evidencethat H-Y is preferentially expressed on Y-bearing sperm (e.g.Hendricksen et al. 1993, Mol. Reprod. Devel. 35:189) and, in a review,Windsor et al. (1993, Reprod. Fert. Dev. 5:155) have concluded that nodifferences between the two classes of sperm can be detectedimmunologically.

Another method recently described as showing utility for sexpre-selection involves the use of Fluorescence Activated Cell Sorting(FACS) for sorting sperm based on the reduced amount of DNA in Y spermas opposed to X sperm due to the small mass of the Y chromosome. Thedifference in DNA content between X and Y sperm, ranges from 2.8% inhumans and 4.0% in most livestock, to 12.5% in voles (Gillis, 1995).See, e.g. Rath et al., 1999, J. Anim. Sci. 77:3346-3352; Welch andJohnson, 1999, Theriogenology 52:1343-1352; Fugger et al., 1998, HumanReprod. 13: 2367-2370; Cran et al., 1995, Vet. Rec. 135: 495-496; Seidelet al., 1997, Theriogenology 48: 1255-1265.

FACS sorting, following by insemination, has been shown to work inbulls, rams (Johnson and Clark, 1988) and humans (Johnson et al., 1993).In spite of these successes, this technique is limited by three factors.First, it requires the sophisticated operation of expensive machines.Second, the reagents used to fluorescently label the DNA and the near UVlight used to detect the dyes may lead to chromosomal damage and/ormutations. Third, this technique has a poor yield. Progress in thesetechniques has recently been summarized in review articles by Reubinoffand Schenker (1996) and Botcham et al (1997).

In another example, which combines sorting based on DNA content,followed by immunological selection, Spaulding, (U.S. Pat. Nos.5,021,244 and 5,346,990, and 5,660,997) first sorted sperm into enrichedX- and Y-chromosome bearing preparations via DNA content and cellsorting techniques. Spaulding then used the sorted sperm to screen forsex-specific sperm proteins and then proceeded to predict the use of thesex-specific protein for raising antibodies to allow purification of thesperm population to either X-chromosome bearing or Y-chromosome bearingpopulations.

WO 01/47353 proposes methods by which expression of a transgene insertedinto a sex chromosome might alter the sex ratio of offspring.

The dairy industry demands a large number of females cows for theproduction of milk, and currently male calves, except those necessaryfor breeding, are culled. Similarly, for the production of beef, malecattle are preferred. In spite of recent progress in techniques forsorting male sperm (Y) from female sperm (X), the techniques still lackthe robustness needed for routine use for the commercial production oflivestock. One reason is that the techniques available are difficult touse to produce the large numbers of viable spermatozoa required for usein the production of livestock. Also, some of the techniques carry withthem the threat of creating mutations while sorting sperm. Thus, thereremains a need in the art for methods and materials permitting the sexpre-selection of offspring.

SUMMARY OF THE INVENTION

The present invention discloses a robust technique for producing sementhat is enriched for active sperm containing either the X chromosome orthe Y chromosome. Because cows of reproductive age normally will givebirth to only a single calf per year, which will randomly either be maleor female, the ability to pre-select the sex of an offspring isparticularly advantageous for the dairy and meat industries. However, inthe agricultural industry generally, methods for sex selection could beused to upgrade the nutritional characteristics and quantities ofanimals produced. Accurate selection of the sex of the offspring couldallow the birth of many genetically superior animals of a single sex asoffspring of one genetically desirable parent. Thereby, the desirablegenetic characteristics of the parent animals can be propagated withmuch greater velocity than is possible in nature. The ability toincrease the reproductive capacity of genetically prized animals,especially dairy cattle, may be a key to solving the hunger problemwhich exists in many countries today by allowing a more efficient use ofavailable resources.

In a first aspect, this invention relates to animals in which one ormore transgenes are incorporated into either the X or Y chromosome, andhence into those sperm cells containing a specific sex chromorome, ofthe transgenic animal. Preferably, the transgene(s) is (are) under thecontrol of a promoter region and/or an enhancer region which is capableof conferring haploid-specific expression to the coupled trausgene. Inthese embodiments, the semen produced by the transgenic animal can beenriched for sperm of a given-sex by expression of the transgene.

Transgenes useful for this invention include genes that encode a geneproduct which is toxic for a haploid cell when expressed in cis, e.g.,suicide genes such as pertussis toxin or the immunoglobulin heavy chainbinding protein (BiP); alternatively, gene products that allow forsurvival in cis when the sperm cell is exposed to a selective agent maybe employed. The term “in cis” is defined hereinafter. In otherembodiments, the gene may encode an antisense construct capable ofblocking the expression of a gene essential for the continued viabilityor function of the sperm.

The only requirement of the transgene(s) used in the instant inventionis that they may be expressed in a haploid-specific manner, and thattransgene expression results in enhanced production of offspring havingthe selected sex. The transgenes of the instant invention need notresult in the death of the haploid cells in which it is expressed,however, in order to enrich for sperm of a selected sex. For example, agene may prevent induction of pregnancy by a haploid cell, for exampleby preventing fusion of a sperm with an oocyte, or by reducing orpreventing motility. Even a minor change in fitness, resulting from thepresence of one or more transgenes, may result in enhanced production ofoffspring having the selected sex. See, e.g., Ellison et al., Mol.Reprod. Dev. 55: 249-55 (2000).

The transgenes of the instant invention may also encode gene productsthat allow the haploid cells expressing the gene to be detected by adetection method, e.g., optically. Genes which can be detected opticallyinclude the Green Fluorescent Protein (GFP) (Tsien, 1998, Annu. Rev.Biochem. 67:509-44), drFP83 and the ES mutant (Terskikh, et al., 2000,Science 290:1585-1588).

Finally, the transgenes of the instant invention may encode geneproducts that make a haploid cell apparent to an in vivo immuneresponse. For example, sex chromosome-specific immune infertility may beproduced by immunizing an animal against a transgene product expressedin a sex chromosome-specific and haploid-specific manner. Such immunitymay be created in either a male or a female, resulting in enhancedproduction of offspring of the selected sex. See, e.g. Tsuji et al., J.Reprod. Immunol. 46: 31-8 (2000); Mahmoud et al., Andrologica 28: 191-6(1996).

The term “haploid cell” as used herein refers to cells that contain asingle set of unpaired chromosomes. In animals, cells that give rise togametes (i.e., sperm and eggs) undergo meiotic division, whereby adiploid cell divides into four haploid cells. In males, a diploid cellcontains both an X and a Y chromosome, referred to herein as “sexchromosomes.” Each haploid cell contains only one sex chromosome. Theterm “haploid cell” can preferably refer to the following cells producedby a male animal: primary spermatocytes (produced in the first meioticdivision); secondary spermatocytes (produced in the second meioticdivision); spermatids; differentiating spermatids; and spermatozoa. Theterm “haploid cell” can also refer to cells produced by a female animal,e.g., oocytes and eggs.

The term “transgenic” as used herein refers to a cell or an animal thatcomprises heterologous deoxyribonucleic acid (DNA). Methods forproducing transgenic cells and animals are well known to the ordinarilyskilled artisan. See, e.g., Mitani et al., 1993, Trends Biotech, 11:162-166; U.S. Pat. No. 5,633,067, “Method of Producing a TransgenicBovine or Transgenic Bovine Embryo,” DeBoer et al., issued May 27, 1997;U.S. Pat. No. 5,612,205, “Homologous Recombination in Mammalian Cells,”Kay et al, issued Mar. 18, 1997; and PCT publication WO 93/22432,“Method for Identifying Transgenic Pre-Implantation Embryos;” Kereso etal., 1996, Chromosome Research 4: 226-239; Holló et al., 1996,Chromosome Research 4: 240-247; U.S. Pat. No. 6,025,155, and U.S. Pat.No. 6,077,697; all of which are incorporated by reference herein intheir entirety, including all figures, drawings, and tables.

The term “heterologous DNA” refers to DNA having (1) a different nucleicacid sequence than DNA sequences present in cell nuclear DNA; (2) asubset of DNA having a nucleotide sequence present in cell nuclear DNA,where the subset exists in different proportions in the heterologous DNAthan in the cell nuclear DNA; (3) a DNA sequence originating fromanother organism species than the species from which cell nuclear DNAoriginates; and/or (4) a different nucleic acid sequence than DNAsequences present in cell mitochondrial DNA. An artificial chromosomepresent in a transgenic cell can comprise heterologous DNA. HeterologousDNA can encode multiple types of recombinant products, as definedhereafter.

The term “different nucleic acid sequence” as used herein refers tonucleic acid sequences that are not substantially similar. The term“substantially similar” as used herein in reference to nucleic acidsequences refers to two nucleic acid sequences having preferably 80% ormore nucleic acid identity, more preferably 90% or more nucleic acididentity or most preferably 95% or more nucleic acid identity. Nucleicacid identity is a property of nucleic acid sequences that measurestheir similarity or relationship. Identity is measured by dividing thenumber of identical bases in the two sequences by the total number ofbases and multiplying the product by 100. Thus, two copies of exactlythe same sequence have 100% identity, while sequences that are lesshighly conserved and have deletions, additions, or replacements have alower degree of identity. Those of ordinary skill in the art willrecognize that several computer programs are available for performingsequence comparisons and determining sequence identity.

A “transgenic animal” is an animal having cells that contain DNA whichhas been artificially inserted into a cell, which DNA becomes part ofthe genome of the animal which develops from that cell. Preferredtransgenic animals are mammals, most preferably non-human primates,mice, rats, ungulates (including cows, pigs, horses, goats, and sheep),dogs and cats. Preferably, a transgenic animal expresses one or moregene products in a haploid-specific manner. Additionally, preferredsites of integration of a heterologous DNA in a transgenic animal of theinstant invention include the Y chromosome and the X chromosome.

Numerous methods are well known in the art for producing transgenicanimals. For example, a nucleic acid construct according to theinvention can be injected into the pronucleus of a fertilized egg beforefusion of the male and female pronuclei, or injected into the nucleus ofan embryonic cell (e.g., the nucleus of a two-cell embryo) following theinitiation of cell division (Brinster et al., Proc. Nat. Acad. Sci. USA82:4438-4442, 1985). Alternatively, embryos can be infected withviruses, especially retroviruses, modified to carry nucleic acidconstructs according to the invention, or other gene delivery vehicles.In particularly preferred embodiments, transgenic animals can beproduced by nuclear transfer using a transgenic nuclear donor cell.Nuclear transfer methods are well known to the ordinarily skilledartisan, and are described in detail hereinafter. See, e.g., U.S. Pat.No. 6,107,543; U.S. Pat. No. 6,011,197; Proc. Nat'l. Acad. Sci. USA 96:14984-14989 (1999); Nature Genetics 22: 127-128 (1999); Cell & Dev. Diol10: 253-258 (1999); Nature Biotechnology 17: 456-461 (1999); Science289: 1188-1190 (2000); Nature Biotechnol. 18: 1055-1059 (2000); Nature407: 86-90 (2000).

The term “transgene” refers to the heterologous DNA included in atransgenic cell or animal. The transgene may refer to the codingsequence or it may also refer to the coding sequence plus additional 5′and 3′ DNA sequences necessary for the proper expression of thetransgene. A cell may contain multiple transgenes, which may or may notbe identical to one another.

The term “expression” as used herein refers to the production of theprotein encoded by a transgene useful in the invention from a nucleicacid vector containing protease genes within a cell. The nucleic acidvector is transfected into cells using well known techniques in the artas described herein. The nucleic acid vector is preferably integratedinto the genome of the host.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene sequence expression. Theprecise nature of the regulatory regions needed for gene sequenceexpression may vary from organism to organism, but shall in generalinclude a promoter region which directs the initiation of RNAtranscription. Such regions will also normally include those5′-non-coding sequences involved with initiation of transcription andtranslation, such as the TATA box, capping sequence, CAAT sequence, andthe like.

The term “promoter” as used herein, refers to nucleic acid sequenceneeded for gene sequence expression. Promoter regions vary from organismto organism, but are well known to persons skilled in the art fordifferent organisms. For example, in prokaryotes, the promoter regioncontains both the promoter (which directs the initiation of RNAtranscription) as well as the DNA sequences which, when transcribed intoRNA, will signal synthesis initiation. Such regions will normallyinclude those 5′-non-coding sequences involved with initiation oftranscription and translation, such as the TATA box, capping sequence,CAAT sequence, and the like. In preferred embodiments, a promoter issex-specific, and/or sperm-specific, -and/or inducible. A particularlypreferred promoter is the protamine promoter.

The term “sex chromosome-specific expression” refers to expression of agene product in cells with a specific sex chromosome. Particularlypreferred is sex chromosome-specific expression in haploid cells, which,by definition, contain only a single sex chromosome. Sexchromosome-specific expression of a gene can be achieved by insertingthe gene to be expressed into the specific sex chromosome. In preferredembodiments, a gene is rendered X chromosome-specific by its operableincorporation into the X chromosome. In these embodiments, only haploidcells that contain an X chromosome will exhibit expression of the geneproduct. In a similar fashion, a gene may be rendered Ychromosome-specific by its operable incorporation into the Y chromosome.

The term “haploid-specific expression” refers to expression of a geneproduct only by haploid cells, such as spermatozoa, spermatids, etc. Thegene product may be expressed during assembly, during spermatogenesis,or after at any time prior to fertilization. In particularly preferredembodiments, a gene that is expressed in a haploid-specific fashion isalso expressed in a sex chromosome-specific fashion.

The transgenes of the instant invention may also be configured andarranged to confer “tissue-specific” expression on the transgene. Thatis, the expression of the transgene may take place only in specific bodytissue(s) of the transgenic animal. Particularly preferred aretransgenes that are expressed only in the testis or only in the ovary ofthe transgenic animal.

The term “specific expression” refers to gene expression that ispredominantly localized to a desired cell type. Such expression may be“leaky,” i.e., there may be some ectopic expression of the gene inundesired cell types, but the predominant expression may still be in thespecific cell type. In preferred embodiments, “specific expression”refers to a gene that is expressed 5-fold higher, 10-fold higher,20-fold higher, 50-fold higher, and 100-fold higher or more in thedesired cell type when compared to expression in undesired cells.

Regulatory sequences that may provide for haploid-specific expressionand/or tissue-specific expression are well known to the skilled artisan.See, e.g., Yamanaka et al., Biol. Reprod. 62: 1694-1701 (2000);Westbrook et al., Biol. Reprod. 63: 469-81 (2000); Tosaka et al., GenesCells 5: 265-76 (2000); Reddi et al., Biol. Reprod. 61: 1256-66 (1999);Nayernia et al., Biol. Reprod. 61: 1488-95 (1999); Mohapatra et al.,Biochem. Biophys. Res. Comm. 244: 540-5 (1998); Herrada et al., J. CellSci. 110: 1543-53 (1997); Rodriguez et al., J. Androl. 21: 414-20(2000); and Lee et al., Biol. Chem. Hoppe Seyler 368: 807-11 (1987). Inpreferred embodiments, the gene that is expressed in a haploid-specificmanner is under the control of the promoter of the protamine gene. See,e.g., Queralt and Olivia, Gene 133: 197-204 (1993).

In certain preferred embodiments, the transgene is capable of killinghaploid cells in which it is expressed (“in cis”) and not in cells notexpressing the transgene; while in other preferred embodiments, thetransgene is capable of functionally disabling haploid cells in cis whenexpressed.

The term “killing haploid cells” refers to the ability of one or moreexpressed gene products to kill a haploid when expressed. The gene(s)may kill the haploid either directly though the activity of one or moreexpressed proteins, or indirectly, via metabolizing an exogenouslysupplied compound to produce a toxic product or by failing to metabolizea toxic chemical supplied exogenously. In preferred embodiments, thegene product(s) are expressed in a haploid-specific manner; in otherembodiments, the gene product(s) are expressed in an inducible fashion.Particularly preferred as a gene to kill haploid cells is theimmunoglobulin heavy chain binding protein (BiP) gene, mutations ofwhich have been shown to exhibit dominant negative effects in cells.See, e.g., Hendershot et al., Proc. Natl. Acad. Sci. USA 93: 5269-74(1996).

The skilled artisan will recognize that expression of a gene may alsorender haploid cells in which it is expressed viable in the presence ofa molecule that would ordinarily kill or disable the cells. Such astrategy is often used, e.g., by inserting antibiotic resistance genesinto cells, then killing those cells that do not express the resistancegene by contacting the cells with an antibiotic.

The term “disabling haploid cells” refers to the ability of one or moreexpressed gene products to prevent the proper functioning of a haploidcell when expressed, without killing the cell. Genes which may disablehaploid cells include, but are not limited to, (1) proteins that disturbionic gradients by forming pores in the membranes of a cell, bothextracellular and intracellular, (2) proteins that interfere with themotility of sperm, e.g., by binding to microtubules, by affectingprotein tyrosine kinases, etc., (3) enzymes capable of degrading DNAsuch as those involved in apoptosis, (4) proteins that are directlytoxic to the cell, (5) enzymes that produce a compound which is toxic tothe cell when supplied with an exogenous metabolite, and (6) proteinsthat affect energy metabolism. The term “disabling” can also refer toacting upon a haploid cell so as to reduce or destroy its mobility, todisrupt or degrade its DNA so as to block the ability of the DNA to beused in creating a viable offspring, or to prevent it from binding toand combining with another haploid cell (i.e., participating infertilization). See, e.g., Uma Devi et al., Andrologia 32: 95-106(2000); Jelks et al., Reprod. Toxicol. 15: 11-20 (2001); Jones &Bavister, J. Androl. 21: 616-24 (2000).

In yet another preferred embodiment, the transgene is a marker gene thatencodes a product which can be detected and used as a basis for sortinghaploid cells. Preferably, the protein encoded allows for opticaldetection. Such a protein can be a fluorescent protein.

The term “marker gene” refers to a gene which can be used to physicallyseparate cells expressing this marker from cells not expressing thismarker. One such gene is green fluorescent protein.

The term “sort” refers to the process of creating two populations ofhaploid cells with one population enriched for cells containing aspecific sex chromosome. This term can refer to FACS sorting, atechnique which is familiar to one skilled in the art. The term may alsoencompass others means of creating a population of cells enriched for aspecific sex chromosome such as affinity purification by a marker foundon the surface of cells, or some other means of selection.

While the gene(s) described above can be expressed in the final haploidcell types produced by males and females (i.e., spermatozoa and eggs),the skilled artisan will understand that a population of these finalcells enriched for cells containing a specific sex chromosome can beobtained by expressing the gene(s) in precursors to those final cells.For example, one or more transgenes can be expressed in primaryspermatocytes that kill only those cells containing the transgene(s). Asa result, only those cells not expressing the gene can mature intospermatozoa.

The term “X sperm” refers to a sperm or spermatozoa which includes onlyan X sex chromosome. Such cells may also be referred to as X-chromosomesperm or an X-chromosome-bearing sperm. Similarly, the term “Y sperm”refers to a sperm or spermatozoa which includes only a Y sex chromosome.Such cells may also be referred to as Y-chromosome sperm or anY-chromosome-bearing sperm.

The term “enriched” means both purifying in an numerical sense andpurifying in a functional sense. “Enriched” does not imply that thereare no undesired cells are present, just that the relative amount of thecells of interest have been significantly increased in either a numericor functional sense. First, by the use of the term “enriched” inreferring to haploid cells in a numerical sense is meant that thedesired cells constitute a significantly higher fraction (2- to 5-fold)of the total haploid cells present. This would be caused by a person bypreferential reduction in the amount of the other haploid cells present.

The term “enriched” in reference to haploid cells may also mean that thespecific cells desired constitute a significantly higher fraction (2- to5-fold) of the total, functional haploid cells present. This would becaused by a person by preferential reduction in the amount of functionalundesired cells. “Enriched” may also mean that one population of haploidcells is at some competitive disadvantage in comparison to anotherpopulation. For example, a small decrease in fitness of, say, Xchromosome-bearing sperm may dramatically reduce their ability tocompete with Y chromosome-bearing sperm to fertilize an ovum.

The term “significant” is used to indicate that the level of increase isuseful to the person making such an increase, and generally means anincrease relative to the other of haploid cells of about at least2-fold, more preferably at least 5- to 10-fold or even more. That is,the term is meant to cover only those situations in which a person hasintervened to elevate the proportion of the desired haploid cells.

The term “functional sperm” means sperm that are capable of fertilizingova. In preferred embodiments, a functional sperm is motile, capable ofbinding to ova, capable of transferring their DNA to the ova, andcontain undamaged DNA. The skilled artisan will understand that not allof these characteristics are required for a sperm to function, however.For example, non-motile sperm can be directly injected into eggs toinitiate fertilization.

In preferred embodiments, a transgenic animal is a mammal, mostpreferably an ungulate. Particularly preferred transgenic animals areselected from the group consisting of a bovid, ovid, suid, equid,caprid, and cervid.

The term “mammalian” as used herein refers to any animal of the classMammalia. Preferably, a mammal is a placental, a monotreme and amarsupial. Most preferably, a mammalis a canid, felid, murid, leporid,ursid, mustelid, ungulate, ovid, suid, equid, bovid, caprid, cervid, anda human or non-human primate.

The term “canid” as used herein refers to any animal of the familyCanidae. Preferably, a canid is a wolf, a jackal, a fox, and a domesticdog. The term “felid” as used herein refers to any animal of the familyFelidae. Preferably, a felid is a lion, a tiger, a leopard, a cheetah, acougar, and a domestic cat. The term “murid” as used herein refers toany animal of the family Muridae. Preferably, a murid is a mouse and arat. The term “leporid” as used herein refers to any animal of thefamily Leporidae. Preferably, a leporid is a rabbit. The term “ursid” asused herein refers to any animal of the family Ursidae. Preferably, aursid is a bear. The term “mustelid” as used herein refers to any animalof the family Mustelidae. Preferably, a mustelid is a weasel, a ferret,an otter, a mink, and a skunk. The term “primate” as used herein refersto any animal of the Primate order. Preferably, a prlimate is an ape, amonkey, a chimpanzee, and a lemur.

The term “ungulate” as used herein refers to any animal of thepolyphyletic group formerly known as the taxon Ungulata. Preferably, anungulate is a camel, a hippopotamus, a horse, a tapir, and an elephant.Most preferably, an ungulate is a sheep, a cow, a goat, and a pig.Especially preferred in the bovine species are Bos taurus, Bos indicus,and Bos buffaloes cows or bulls. The term “ovid” as used herein refersto any animal of the family Ovidae. Preferably, an ovid is a sheep. Theterm “suid” as used herein refers to any animal of the family Suidae.Preferably, a suid is a pig or a boar. The term “equid” as used hereinrefers to any animal of the family Equidae. Preferably, an equid is azebra or an ass. Most preferably, an equid is a horse. The term “bovid”as used herein refers to any animal of the family Bovidae. Preferably,an bovid is an antelope, an oxen, a cow, and a bison. The term “caprid”as used herein refers to any animal of the family Caprinae. Preferably,a caprid is a goat. The term “cervid” as used herein refers to anyanimal of the family Cervidae. Preferably, a cervid is a deer.

In certain embodiments, this invention relates to animals in which oneor more transgenes capable of being expressed in a haploid-specificmanner in cells is incorporated into the genome, and hence the haploidcells, of the transgenic animal. This transgene can be under the controlof a promoter region and/or an enhancer region which is capable ofconferring sex chromosome-specific expression on the coupled transgene;and this transgene can also under the control of a promoter regionand/or an enhancer region which only allows expression of its operablylinked gene when provided specific inducing agent.

The term “inducible” refers to a promoter which is only active in thepresence of specific inducing agent. Preferably the inducing agent issupplied exogenously. The inducing factor may require binding to othercellular components in order to achieve the intended result ofincreasing transcription. Examples of inducible promoters are well knownto those skilled in the art. The exogenous inducing agent may be givento the animal producing the sperm, or it may be incubated with isolatedsperm. The inducing agent may also be produced endogenously by theanimal from which the enriched sperm is to be isolated.

For instance, an inducible promoter, such as the IL-8 promoter that isresponsive to TNF or another cytokine, can be employed. Other examplesof suitable inducible promoter systems include, but are not limited to,the metallothionine inducible promoter system, the bacterial lacZYAexpression system, the tetracycline expression system, and the T7polymerase system. Further, promoters that are selectively activated atdifferent developmental stages (e.g., globin genes are differentiallytranscribed in embryos and adults) can be employed. Still otherpossibilities include the use of a glucocorticoid response element or atetracycline response element.

Construction of an exogenous nucleic acid operably linked to a promoteris also well within the skill of the art (See, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, (2d ed. 1989) which ishereby incorporated by reference herein in its entirety including anyfigures, tables, or drawings.). With respect to the transfer andexpression of exogenous nucleic acids according to the presentinvention, one skilled in the art is aware that different geneticsignals and processing events control levels of nucleic acids andproteins/peptides in a cell, including transcription, MRNA translation,and post-transcriptional processing. Transcription of DNA into RNArequires a functional promoter.

Protein expression is dependent on the level of RNA transcription whichis regulated by DNA signals. Similarly, translation of MRNA requires, atthe very least, an AUG initiation codon, which is usually located within10 to 100 nucleotides of the 5′ end of the MRNA. Sequences flanking theAUG initiator codon have been shown to influence its recognition byeukaryotic ribosomes, with conformity to a perfect Kozak consensussequence resulting in optimal translation (see, e.g., Kozak, J. Molec.Biol., 1987, 196:947-950). Also, successful expression of an exogenousnucleic acid in a cell can require post-translational modification of aresultant protein. Thus, production of a recombinant protein can beaffected by the efficiency with which DNA (or RNA) is transcribed intomRNA, the efficiency with which mRNA is translated into protein, and theability of the cell to carry out post-translational modification. Theseare all factors of which one skilled in the art is aware and is capableof manipulating using standard means to achieve the desired end result.

Along these lines, to optimize protein production, preferably thetransgenic nucleic acid sequence further comprises a polyadenylationsite following the coding region of the transgenic nucleic acid. Also,preferably all the proper transcription signals (and translationsignals, where appropriate) will be correctly arranged such that thetransgenic nucleic acid sequence will be properly expressed in the cellsinto which it is introduced. If desired, the transgenic nucleic acidalso can incorporate splice sites (i.e., splice acceptor and splicedonor sites) to facilitate mRNA production. Moreover, if the transgenicnucleic acid sequence encodes a protein, which is a processed orsecreted protein or functions in intracellular organelles, such as amitochondria or the endoplasmic reticulum, preferably the transgenicnucleic acid further comprises the appropriate sequences for processing,secretion, intracellular localization, and the like. Such sequences andsignals are well known to those skilled in the art.

The term “non-functional” in reference to a spermatozoa refers to cellsthat are no longer capable of fertilizing an ovum. This may be due todeficiencies in chromosome integrity, motility, or composition of theouter membrane.

In yet another aspect, the invention relates to methods for producing apopulation of haploid cells which are enriched for cells containing aspecific sex chromosome, either the X or the Y, where the haploid cellsare harvested from an animal comprising one or more transgenes that arecapable of killing or disabling cells in cis when expressed. Thetransgene(s) are preferably under the control of a promoter which isonly active in sperm containing a specific sex chromosome. In preferredembodiments, this promoter is active only in sperm containing a Xchromosome; and this promoter is active only in sperm containing a Ychromosome. The promoter of the invention is also only active in haploidcells. The transgene then is allowed to act to kill or disable haploidcells containing the selected chromosome. Viable and/or functionalhaploid cells may be optionally purified away from the non-functionalsperm by techniques known to those skilled in the art.

In still another aspect, the invention relates to methods for producinga population of haploid cells which are enriched for cells containing aspecific sex chromosome, either the X or the Y, where the haploid cellsare harvested from an animal comprising one or more transgenes which arecapable of killing or disabling cells in cis when expressed, where thepromoter of the invention is only active in the presence of an inducingagent. In certain preferred embodiments, this promoter is active only inhaploid cells containing a X chromosome, and this promoter is activeonly in haploid cells containing a Y chromosome. The cells are exposedto an inducing agent, and the promoter region of the transgene(s) thenacts to express the transgene(s) in cells containing one sex chromsomebut not the other. The haploid cells may be exposed in vivo, either inthe source animal or in the maternal host, or they may be exposed invitro. The transgene then acts to kill or disable those haploid cellscontaining the selected chromosome.

In the foregoing aspects, one or more transgenes may optionally be usedwhich do not kill or disable the haploid cells expressing thetransgene(s), but rather causes the expression of a marker gene. Thisexpressed marker may then be used to sort X-chromosome-bearing cellsfrom Y-chromosome-bearing cells by techniques well known to thoseskilled in the art.

In another aspect of the invention, the invention relates to methods forproducing an animal using a population of spermatozoa that is enrichedfor cells containing a specific sex chromosome, either the X or the Y.The offspring produced will thus be primarily of the selected sex. Inpreferred embodiments, if the fertilization of ova using selected spermhas been conducted in vitro, the resultant embryo is transplanted into amaternal host.

In yet another aspect, the invention relates to recombinant nucleicacids arranged and configured for performing the aspects describedabove, whether in vitro or in a cell or an organism. The transgenes ofthe instant invention are preferably comprised in the transgenic animalsof the invention. The recombinant nucleic acids can alternativelycontain a transcriptional initiation region functional in a cell, asequence complementary to an RNA sequence encoding a proteasepolypeptide and a transcriptional termination region functional in acell. Specific vectors and host cell combinations are discussed herein.

The present invention also relates to cells and/or organisms thatcontain the foregoing transgenic nucleic acid molecules incorporatedinto the genome, and thereby which are capable of expressing apolypeptide or other gene of interest. A cell is said to be “altered toexpress a desired polypeptide or other gene of interest” when the cell,through genetic manipulation, is made to produce a protein or other geneof interest which it normally does not produce or which the cellnormally produces at lower levels. One skilled in the art can readilyadapt procedures for introducing and expressing either genomic, cDNA, orsynthetic sequences into eukaryotic cells.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide or other gene of interest if it containsnucleotide sequences which contain transcriptional and translationalregulatory information and such sequences are “operably linked” tonucleotide sequences which encode the polypeptide. An operable linkageis a linkage in which the regulatory DNA sequences and the DNA sequencesought to be expressed are connected in such a way as to permit genesequence expression. The precise nature of the regulatory regions neededfor gene sequence expression may vary from organism to organism, butshall in general include a promoter region and other 5′-non-codingsequences involved with initiation of transcription and translation,such as the TATA box, capping sequence, CAAT sequence, and the like.

Two DNA sequences (such as a promoter region sequence and a sequenceencoding the gene of interest) are said to be operably linked if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the promoter region sequence to direct the transcription of agene sequence encoding the gene of interest, or (3) interfere with theability of the gene sequence of the gene of interest to be transcribedby the promoter region sequence. Thus, a promoter region would beoperably linked to a DNA sequence if the promoter were capable ofeffecting transcription of that DNA sequence. Thus, to express a geneencoding the gene of interest, transcriptional and translational signalsrecognized by an appropriate host are necessary.

The present invention encompasses the expression of a gene encoding thegene of interest (or a functional derivative thereof) in eukaryoticcells.

The selection of control sequences, expression vectors, transformationmethods, and the like, are dependent on the type of host cell used toexpress the gene, and their selection is well within the skill of theartisan.

As used herein, “cell”, “cell line”, and “cell culture” may be usedinterchangeably and all such designations include progeny. Thus, thewords “transformants” or “transformed cells” include the primary subjectcell and cultures derived therefrom, without regard to the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.However, as defined, mutant progeny have the same functionality as thatof the originally transformed cell.

The term “vector” relates to a single or double-stranded circularnucleic acid molecule that can be transfected into cells and replicatedwithin or independently of a cell genome. A circular double-strandednucleic acid molecule can be cut and thereby linearized upon treatmentwith restriction enzymes. An assortment of nucleic acid vectors,restriction enzymes, and the knowledge of the nucleotide sequences cutby restriction enzymes are readily available to those skilled in theart. A nucleic acid molecule encoding a protease can be inserted into avector by cutting the vector with restriction enzymes and ligating thetwo pieces together. Preferred vectors are those designed for performing“gene targeting” procedures. See, e.g., U.S. Pat. Nos. 6,090,554,6,069,010, 5,792,663, and 5,789,215, each of which is herebyincorporated by reference in its entirety, including all tables,figures, and claims.

The term “transfecting” defines a number of methods to insert a nucleicacid vector or other nucleic acid molecules into a cellular organism.These methods involve a variety of techniques, such as treating thecells with high concentrations of salt, an electric field, detergent, orDMSO to render the outer membrane or wall of the cells permeable tonucleic acid molecules of interest or use of various viral transductionstrategies.

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus,cytomegalovirus, simian virus, or the like, where the regulatory signalsare associated with a particular gene sequence which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, and the like, may be employed.Transcriptional initiation regulatory signals may be selected whichallow for repression or activation, so that expression of the genesequences can be modulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

Expression of the transgenes of the invention in eukaryotic hostsrequires the use of eukaryotic regulatory regions. Such regions will, ingeneral, include a promoter region sufficient to direct the initiationof RNA synthesis. Preferred eukaryotic promoters include, for example,the promoter of the mouse metallothionein I gene sequence (Hamer et al.,J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus(McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist etal., Nature (London) 290:304-31, 1981); and the yeast gal4 gene sequencepromoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975,1982; Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984).

Translation of eukaryotic mRNA is initiated at the codon which encodesthe first methionine. For this reason, it is preferable to ensure thatthe linkage between a eukaryotic promoter and a DNA sequence whichencodes the gene of interest (or a functional derivative thereof) doesnot contain any intervening codons which are capable of encoding amethionine (i.e., AUG). The presence of such codons results either inthe formation of a fusion protein (if the AUG codon is in the samereading frame as the protease of the invention coding sequence) or aframe-shift mutation (if the AUG codon is not in the same reading frameas the protease of the invention coding sequence).

A nucleic acid molecule encoding the gene of interest and an operablylinked promoter may be introduced into a recipient host cell either as anonreplicating DNA or RNA molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule.Permanent expression will occur through the integration of theintroduced DNA sequence into the host chromosome.

A vector may be employed which is capable of integrating the desiredgene sequences into the host cell chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes can be selected byalso introducing one or more markers which allow for selection of hostcells which contain the expression vector. The marker may provide forprototrophy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene sequence can either be directly linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection. Additional elements may also be needed for optimalsynthesis of MRNA. These elements may include splice signals, as well astranscription promoters, enhancers, and termination signals. cDNAexpression vectors incorporating such elements include those describedby Okayama (Mol. Cell. Biol. 3:280-289, 1983).

The introduced nucleic acid molecule can be incorporated into a plasmidor viral vector capable of autonomous replication in the recipient host.Any of a wide variety of vectors may be employed for this purpose.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Once the vector or nucleic acid molecule containing the construct(s) hasbeen prepared for expression, the DNA construct(s) may be introducedinto an appropriate host cell by any of a variety of suitable means,i.e., transformation, transfection, conjugation, protoplast fusion,electroporation, particle gun technology, calciumphosphate-precipitation, direct microinjection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene(s) results in the production of the geneof interest, or fragments thereof. This can take place in thetransformed cells as such, or following the induction of these cells todifferentiate (for example, by administration of bromodeoxyuracil toneuroblastoma cells or the like). A variety of incubation conditions canbe used to form the peptide of the present invention. The most preferredconditions are those which mimic physiological conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 shows, in schematic form, spermatogenesis, i.e., the productionof haploid cells from diploid precursors that occurs in male animals.

FIG. 2 shows, in schematic form, an exemplary procedure for producing atransgenic animal of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes materials and methods for producingsemen that is enriched for active sperm containing either the Xchromosome or the Y chromosome, by producing transgenic animals thatexpress one or more genes in a sex chromosome-specific and/orhaploid-specific manner. As discussed above, the ability to pre-selectthe sex of an offspring is particularly advantageous in the agriculturalindustry. By allowing for the selection of a specific population ofhaploid cells, the materials and methods described herein can facilitatethis sex pre-selection.

I. Transgenic Cells and Animals

A. General Methods

Materials and methods readily available to a person of ordinary skill inthe art can be applied to produce transgenic cells and animals. See,e.g., EPO 264 166, entitled “Transgenic Animals Secreting DesiredProteins Into Milk”; WO 94/19935, entitled “Isolation of Components ofInterest From Milk”; WO 93/22432, entitled “Method for IdentifyingTransgenic Pre-implantation Embryos”; WO 95/17085, entitled “TransgenicProduction of Antibodies in Milk;” Hammer et al., 1985, Nature 315:680-685; Miller et al., 1986, J. Endocrinology 120: 481-488; Williams etal., 1992, J. Ani. Sci. 70: 2207-2111; Piedrahita et al., 1998, Biol.Reprod. 58: 1321-1329; Piedrahita et al., 1997, J. Reprod. Fert.(suppl.) 52: 245-254; and Nottle et al, 1997, J. Reprod. Fert. (suppl.)52: 245-254, each of which is incorporated herein by reference in itsentirety including all figures, drawings and tables.

Methods for generating transgenic cells typically include the steps of(1) assembling a suitable DNA construct useful for inserting a specificDNA sequence into the nuclear genome of a cell; (2) transfecting the DNAconstruct into the cells; (3) allowing random insertion and/orhomologous recombination to occur. The modification resulting from thisprocess may be the insertion of a suitable DNA construct(s) into thetarget genome; deletion of DNA from the target genome; and/or mutationof the target genome.

DNA constructs can comprise a gene of interest as well as a variety ofelements including regulatory promoters, insulators, enhancers, andrepressors as well as elements for ribosomal binding to the RNAtranscribed from the DNA construct. DNA constructs can also encoderibozymes and anti-sense DNA and/or RNA, identified previously herein.These examples are well known to a person of ordinary skill in the artand are not meant to be limiting.

Due to the effective recombinant DNA techniques available in conjunctionwith DNA sequences for regulatory elements and genes readily availablein data bases and the commercial sector, a person of ordinary skill inthe art can readily generate a DNA construct appropriate forestablishing transgenic cells using the materials and methods describedherein.

Preferred vectors for use in the present invention are gene targetingvectors, in order to mediate insertion of a gene of interest byhomologous recombination with a site in the host genome. Such vectorstypically include four major elements. A promoter, is linked to, anddrives, the expression of a gene. An Y or X chromosome specific DNAsequence is linked to the promoter/gene elements. The Y or X chromosomespecific sequence is to be used as homologous arms for targeting thevector to the Y or X chromosome, respectively. Finally, a selectionmarker, such as the neomycin-resistance gene, neo (Southern, P. J. &Berg, P. (1982) J Mol Appl Genet 1: 327-341) is typically included.

Preferred elements of the vectors which may be obtained and incorporatedinto the targeting vectors include novel sequence of both the bovine(Lee et al (1987) Biol Chem Hoppe Seyler 368: 131-135; Krawetz et al.,(1988) J Biol Clern 263: 321-326) and porcine (Maier et al., (1988)Nucleic Acids Res 16: 11826) protamine promoters. In addition, apreferred toxic gene, a dominant negative mutant of hamster BiP protein,plus wild-type hamster BiP protein, to disrupt proper protein folding inX- or Y-bearing sperm have been disclosed (Hendershot et al., (1996)Proc Natl Acad Sci U S A 93: 5269-5274; Morris et al., (1997) J BiolChem 272: 4327-4334). Suitable bovine and porcine Y chromosome specificsequences 3′ of the SRY gene (Hacker et al., (1995) Development 121:1603-1614) to be used as homologous arms for gene targeting have alsobeen disclosed.

As described below, complete insertion vectors containing the promoter,gene sequence, selectable marker, and a homologous arm have beenconstructed. A schematic of such a vector is provided below. The vectorcan be linearized by cutting with a restriction enzyme that bisects thehomologous arm prior to transfection to provide a mature insertionvector.

Transfection techniques are well known to a person of ordinary skill inthe art and materials and methods for carrying out transfection of DNAconstructs into cells are commercially available. For example, materialsthat can be used to transfect cells with DNA constructs are lipophilliccompounds such as Lipofectin™, activated polycationic dendrimers such asSuperfect™, LipoTAXI™, and CLONfectin™. Particular lipophillic compoundscan be induced to form liposomes for mediating transfection of the DNAconstruct into the cells. In addition, cationic based transfectionagents that are known in the art can be utilized to transfect cells withnucleic acid molecules (e.g., calcium phosphate precipitation). Also,electroporation techniques known in the art can be utilized totranslocated nucleic acid molecules into cells. Furthermore, particlebombardment techniques known in the art can be utilized to introduceexogenous DNA into cells. Target sequences from a DNA construct can beinserted into specific regions of the nuclear genome by rational designof the DNA construct. These design techniques and methods are well knownto a person of ordinary skill in the art. See, U.S. Pat. No. 5,633,067,“Method of Producing a Transgenic Bovine or Transgenic Bovine Embryo,”DeBoer et al., issued May 27, 1997; U.S. Pat. No. 5,612,205, “HomologousRecombination in Mammalian Cells,” Kay et al., issued Mar. 18, 1997; andPCT publication WO 93/22432, “Method for Identifying TransgenicPre-Implantation Embryos,” each of which is incorporated herein byreference in its entirety, including all figures, drawings, and tables.Once the desired DNA sequence is inserted into the nuclear genome of acell, the location of the insertion region as well as the frequency withwhich the desired DNA sequence has inserted into the nuclear genome canbe identified by methods well known to those skilled in the art.

B. Haploid-Specific Expression

In a preferred embodiment, the protamine promoter can be used toestablish haploid-specific and/or tissue-specific gene expression.Protamine is a small, basic protein that binds to DNA during thecondensation and compaction of the sperm head. Protamine is expressedexclusively in testis, and it is expressed at the haploid stage in roundspermatids following the completion of meiosis. Lee et al., 1987, Biol.Chem. Hoppe Seyler 970: 807-1 1. Regulatory sequences for this gene havebeen found in about 10 species, including bovines. Krawetz et al., 1988,J. Biol. Chem. 263: 321-326; Queralt and Olivia, 1993, Gene 133:197-204.

C. Expression of a Gene Product In Cis

During spermatogenesis, haploid cells at certain stages are joined by“cytoplasmic bridges” that allow sharing of soluble cell contentsbetween adjacent cells. See, e.g., FIG. 1. Thus, if a transgene isselected that produces a freely soluble expression product, and theconstruct chosen allows expression at the stage when these bridges arepresent, the expression product may kill cells containing both sexchromosomes. Therefore, it may be important to select a gene productthat produces its effects only in cis. Such a gene product preferablyexhibits the following characteristics: the ability to exert its effectsin a dominant fashion (i.e., expression of the transgene alone createsthe effect, even against an otherwise wild type expression background);the ability to remain anchored to the matrix of the cell in which it isproduced; and participation in an essential function, so that expressioncauses death or disablement of the cell.

In this regard, a preferred gene, the expression of which can be drivenby the protamine promoter in a haploid-specific fashion, is theimmunoglobulin heavy chain binding protein (BiP). BiP is a HSP 70molecular chaperone. A series of point mutations in a hamster BiPsequence has been shown to inhibit the BiP ATPase activity, resulting ina dominant negative mutant exhibiting disrupted endoplasmic reticulum(ER) function. See, e.g., Hendershot et al., Proc. Natl. Acad. Sci. USA93: 5269-74 (1996). Furthermore, this dominant negative effect can crossspecies; i.e., hamster BiP mutants can disrupt ER function in bovinesfor example.

Expression of such a mutant in spermatids can disrupt the normaldevelopment of spermatozoa. By using gene targeting methods targeted ata Y chromosome-specific or X chromosome-specific intronic sequence, BiPexpression can be made both haploid-specific and sexchromosome-specific.

II. Nuclear Transfer

In preferred embodiments, once a transgene(s) is (are) inserted into thenuclear genome of the totipotent cell, that cell can be used as anuclear donor for cloning a transgenic animal.

Nuclear transfer (NT) techniques are well known to a person of ordinaryskill in the art. See, e.g., U.S. Pat. No. 4,664,097, “NuclearTransplantation in the Mammalian Embryo by Microsurgery and CellFusion,” issued May 12, 1987, McGrath & Solter; U.S. Pat. No. 4,994,384(Prather et al.); U.S. Pat. No. 5,057,420 (Massey et al.); U.S. Pat. No.6,107,543; U.S. Pat. No. 6,011,197; Proc. Nat'l. Acad. Sci. USA 96:14984-14989 (1999); Nature Genetics 22: 127-128 (1999); Cell & Dev. Diol10: 253-258 (1999); Nature Biotechnology 17: 456-461 (1999); Science289: 1188-1190 (2000); Nature Biotechnol. 18: 1055-1059 (2000); andNature 407: 86-90 (2000); each of which is incorporated herein byreference in its entirety, including all figures, tables, and drawings.Exemplary embodiments define a NT technique that provide for efficientproduction of totipotent mammalian embryos.

A. Nuclear Donors

For NT techniques, a donor cell may be separated from a growing cellmass, isolated from a primary cell culture, or isolated from a cellline. The entire cell may be placed in the perivitelline space of arecipient oocyte or may be directly injected into the recipient oocyteby aspirating the nuclear donor into a needle, placing the needle intothe recipient oocyte, releasing the nuclear donor and removing theneedle without significantly disrupting the plasma membrane of theoocyte. Also, a nucleus (e.g., karyoplast) may be isolated from anuclear donor and placed into the perivitelline space of a recipientoocyte or may be injected directly into a recipient oocyte, for example.

B. Recipient Cells

A recipient cell is typically an oocyte with a portion of its ooplasmremoved, where the removed ooplasm comprises the oocyte nucleus.Enucleation techniques are well known to a person of ordinary skill inthe art. See e.g., Nagashima et al., 1997, Mol. Reprod. Dev. 48:339-343; Nagashima et al., 1992, J. Reprod. Dev. 38: 37-78; Prather etal., 1989, Biol. Reprod. 41: 414-418; Prather et al., 1990, J. Exp.Zool. 255: 355-358; Saito et al., 1992, Assis. Reprod. Tech. Andro. 259:257-266; and Terlouw et al., 1992, Theriogenology 37: 309, each of whichis incorporated herein by reference in its entirety including allfigures, tables, and drawings. Cells other than oocytes can also besuccessfully used as recipient cells. See, e.g., Polejaeva et al.,Nature 407(6800): 86-90 (2000).

Oocytes can be isolated from either oviducts and/or ovaries of liveanimals by oviductal recovery procedures or transvaginal oocyte recoveryprocedures well known in the art and described herein. Furthermore,oocytes can be isolated from deceased animals. For example, ovaries canbe obtained from abattoirs and oocytes can be aspirated from theseovaries. The oocytes can also be isolated from the ovaries of a recentlysacrificed animal or when the ovary has been frozen and/or thawed.

Oocytes can be matured in a variety of media well known to a person ofordinary skill in the art. One example of such a medium suitable formaturing oocytes is depicted in an exemplary embodiment describedhereafter. Oocytes can be successfully matured in this type of mediumwithin an environment comprising 5% CO₂ at 39° C. Oocytes may becryopreserved and then thawed before placing the oocytes in maturationmedium. Cryopreservation procedures for cells and embryos are well knownin the art as discussed herein.

Components of an oocyte maturation medium can include molecules thatarrest oocyte maturation. Examples of such components are6-dimethylaminopurine (DMAP) and isobutylmethylxanthine (IBMX). IBMX hasbeen reported to reversibly arrest oocytes, but the efficiencies ofarrest maintenance are quite low. See, e.g., Rose-Hellkant and Bavister,1996, Mol. Reprod. Develop. 44: 241-249. However, oocytes may bearrested at the germinal vesicle stage with a relatively high efficiencyby incubating oocytes at 31° C. in an effective concentration of IBMX.Preferably, oocytes are incubated the entire time that oocytes arecollected. Concentrations of IBMX suitable for arresting oocytematuration are 0.01 mM to 20 mM IBMX, preferably 0.05 mM to 10 mM IBMX,and more preferably about 0.1 mM IBMX to about 0.5 mM IBMX, and mostpreferably 0.1 mM IBMX to 0.5 mM IBMX. In certain embodiments, oocytescan be matured in a culture environment having a low oxygenconcentration, such as 5% O₂, 5-10% CO₂, and 85-90% N₂.

A nuclear donor cell and a recipient oocyte can arise from the samespecies or different species. For example, a totipotent porcine cell canbe inserted into a porcine enucleated oocyte. Alternatively, atotipotent wild boar cell can be inserted into a domesticated porcineoocyte. Any nuclear donor/recipient oocyte combinations are envisionedby the invention. Preferably the nuclear donor and recipient oocyte fromthe same specie. Cross-species NT techniques can be utilized to producecloned animals that are endangered or extinct.

Oocytes can be activated by electrical and/or non-electrical meansbefore, during, and/or after a nuclear donor is introduced to recipientoocyte. For example, an oocyte can be placed in a medium containing oneor more components suitable for non-electrical activation prior tofusion with a nuclear donor. Also, a cybrid can be placed in a mediumcontaining one or more components suitable for non-electricalactivation. Activation processes are discussed in greater detailhereafter.

C. Injection/Fusion

A nuclear donor can be translocated into an oocyte using a variety ofmaterials and methods that are well known to a person of ordinary skillin the art. In one example, a nuclear donor may be directly injectedinto a recipient oocyte. This direct injection can be accomplished bygently pulling a nuclear donor into a needle, piercing a recipientoocyte with that needle, releasing the nuclear donor into the oocyte,and removing the needle from the oocyte without significantly disruptingits membrane. Appropriate needles can be fashioned from glass capillarytubes, as defined in the art and specifically by publicationsincorporated herein by reference.

In another example, at least a portion of plasma membrane from a nucleardonor and recipient oocyte can be fused together by utilizing techniqueswell known to a person of ordinary skill in the art. See, Willadsen,1986, Nature 320:63-65, hereby incorporated herein by reference in itsentirety including all figures, tables, and drawings. Typically, lipidmembranes can be fused together by electrical and chemical means, asdefined previously and in other publications incorporated herein byreference.

Examples of non-electrical means of cell fusion involve incubatingcybrids in solutions comprising polyethylene glycol (PEG), and/or Sendaivirus. PEG molecules of a wide range of molecular weight can be utilizedfor cell fusion.

Processes for fusion that are not explicitly discussed herein can bedetermined without undue experimentation. For example, modifications tocell fusion techniques can be monitored for their efficiency by viewingthe degree of cell fusion under a microscope. The resulting embryo canthen be cloned and identified as a totipotent embryo by the same methodsas those previously described herein for identifying totipotent cells,which can include tests for selectable markers and/or tests fordeveloping an animal.

D. Activation

Methods of activating oocytes and cybrids are known to those of ordinaryskill in the art. See, U.S. Pat. No. 5,496,720, “Parthenogenic OocyteActivation,” Susko-Parrish et al., issued on Mar. 5, 1996, herebyincorporated by reference herein in its entirety including all figures,tables, and drawings.

Both electrical and non-electrical processes can be used for activatingcells (e.g., oocytes and cybrids). Although use of a non-electricalmeans for activation is not always necessary, non-electrical activationcan enhance the developmental potential of cybrids, particularly whenyoung oocytes are utilized as recipients.

Examples of electrical techniques for activating cells are well known inthe art. See, WO 98/16630, published on Apr. 23, 1998, Piedraheidra andBlazer, hereby incorporated herein in its entirety including allfigures, tables, and drawings, and U.S. Pat. Nos. 4,994,384 and5,057,420. Non-electrical means for activating cells can include anymethod known in the art that increases the probability of cell division.Examples of non-electrical means for activating a nuclear donor and/orrecipient can be accomplished by introducing cells to ethanol; inositoltrisphosphate (IP₃); Ca²⁺ ionophore and protein kinase inhibitors suchas 6-dimethylaminopurine; temperature change; protein synthesisinhibitors (e.g., cycloheximide); phorbol esters such as phorbol12-myristate 13-acetate (PMA); mechanical techniques, thapsigargin, andsperm factors. Sperm factors can include any component of a sperm thatenhance the probability for cell division. Other non-electrical methodsfor activation include subjecting the cell or cells to cold shock and/ormechanical stress.

Examples of preferred protein kinase inhibitors are protein kinase A, G,and C inhibitors such as 6-dimethylaminopurine (DMAP), staurosporin,2-aminopurine, sphingosine. Tyrosine kinase inhibitors may also beutilized to activate cells.

Activation materials and methods that are not explicitly discussedherein can be identified by modifying the specified conditions definedin the exemplary protocols described hereafter and in U.S. Pat. No.5,496,720.

F. Manipulation of Embryos Resulting from Nuclear Transfer

An embryo resulting from a NT process can be manipulated in a variety ofmanners. The invention relates to cloned embryos that arise from atleast one NT. Exemplary embodiments of the invention demonstrate thattwo or more NT procedures may enhance the efficiency for the productionof totipotent embryos. Exemplary embodiments indicate that incorporatingtwo or more NT procedures into methods for producing cloned totipotentembryos may enhance placental development. In addition, increasing thenumber of NT cycles involved in a process for producing totipotentembryos may represent a necessary factor for converting non-totipotentcells into totipotent cells. An effect of incorporating two or more NTcycles upon totipotency of resulting embryos is a surprising result,which was not previously identified or explored in the art.

Incorporating two or more NT cycles into methods for cloned totipotentembryos can provide further advantages. Incorporating multiple NTprocedures into methods for establishing cloned totipotent embryosprovides a method for multiplying the number of cloned totipotentembryos.

When multiple NT procedures are utilized for the formation of a clonedtotipotent embryo, oocytes that have been matured for any period of timecan be utilized as recipients in the first, second or subsequent NTprocedures. Additionally, one or more of the NT cycles may be preceded,followed, and/or carried out simultaneously with an activation step. Asdefined previously herein, an activation step may be accomplished byelectrical and/or non-electrical means as defined herein. Exemplifiedembodiments described hereafter describe NT techniques that incorporatean activation step after one NT cycle. However, an activation step mayalso be carried out at the same time as a NT cycle (e.g., simultaneouslywith the NT cycle) and/or an activation step may be carried out prior toa NT cycle. Cloned totipotent embryos resulting from a NT cycle can be(1) disaggregated or (2) allowed to develop further.

If embryos are disaggregated, disaggregated embryonic derived cells canbe utilized to establish cultured cells. Any type of embryonic cell canbe utilized to establish cultured cells. These cultured cells aresometimes referred to as embryonic stem cells or embryonic stem-likecells in the scientific literature. The embryonic stem cells can bederived from early embryos, morulae, and blastocyst stage embryos.Multiple methods are known to a person of ordinary skill in the art forproducing cultured embryonic cells. These methods are enumerated inspecific references previously incorporated by reference herein.

If embryos are allowed to develop into a fetus in utero, cells isolatedfrom that developing fetus can be utilized to establish cultured cells.In preferred embodiments, primordial germ cells, genital ridge cells,and fetal fibroblast cells can be isolated from such a fetus. Culturedcells having a particular morphology that is described herein can bereferred to as embryonic germ cells (EG cells). These cultured cells canbe established by utilizing culture methods well known to a person ofordinary skill in the art. Such methods are enumerated in publicationspreviously incorporated herein by reference and are discussed herein. Inparticularly preferred embodiments, Streptomyces griseus protease can beused to remove unwanted cells from theembryonic germ cell culture.

Cloned totipotent embryos resulting from NT can also be manipulated bycryopreserving and/or thawing the embryos. See, e.g., Nagashima et al.,1989, Japanese J. Anim. Reprod. 35: 130-134 and Feng et al., 1991,T7Theriogenology 35: 199, each of which is incorporated herein byreference in its entirety including all tables, figures, and drawings.Other embryo manipulation methods include in vitro culture processes;performing embryo transfer into a maternal recipient; disaggregatingblastomeres for NT processes; disaggregating blastomeres or inner cellmass cells for establishing cell lines for use in NT procedures; embryosplitting procedures; embryo aggregating procedures; embryo sexingprocedures; and embryo biopsying procedures. The exemplary manipulationprocedures are not meant to be limiting and the invention relates to anyembryo manipulation procedure known to a person of ordinary skill in theart.

III. Development of Cloned Embryos

A. Culture of Embryos In Vitro

Cloning procedures discussed herein provide an advantage of culturingcells and embryos in vitro prior to implantation into a recipientfemale. Methods for culturing embryos in vitro are well known to thoseskilled in the art. See, e.g., Nagashima et al., 1997, Mol. Reprod. Dev.48: 339-343; Petters & Wells, 1993, J. Reprod. Fert. (Suppl) 48: 61-73;Reed et al., 1992, Theriogenology 37: 95-109; and Dobrinsky et al.,1996, Biol. Reprod. 55: 1069-1074, each of which is incorporated hereinby reference in its entirety, including all figures, tables, anddrawings. In addition, exemplary embodiments for media suitable forculturing cloned embryos in vitro are described hereafter. Feeder celllayers may or may not be utilized for culturing cloned embryos in vitro.Feeder cells are described previously and in exemplary embodimentshereafter.

B. Development of Embryos In Utero

Cloned embryos can be cultured in an artificial or natural uterineenvironment after NT procedures and embryo in vitro culture processes.Examples of artificial development environments are being developed andsome are known to those skilled in the art. Components of the artificialenvironment can be modified, for example, by altering the amount of acomponent or components and by monitoring the growth rate of an embryo.

Methods for implanting embryos into the uterus of an animal are alsowell known in the art, as discussed previously. Preferably, thedevelopmental stage of the embryo(s) is correlated with the estrus cycleof the animal.

Embryos from one species can be placed into the uterine environment ofan animal from another species. For example it has been shown in the artthat bovine embryos can develop in the oviducts of sheep. Stice &Keefer, 1993, “Multiple generational bovine embryo cloning,” Biology ofReproduction 48: 715-719. The invention relates to any combination of aporcine embryo in any other ungulate uterine environment. Across-species in utero development regime can allow for efficientproduction of cloned animals of an endangered species. For example, awild boar embryo can develop in the uterus of a domestic porcine sow.

Once an embryo is placed into the uterus of a recipient female, theembryo can develop to term. Alternatively, an embryo can be allowed todevelop in the uterus and then can be removed at a chosen time. Surgicalmethods are well known in the art for removing fetuses from uteri beforethey are born.

EXAMPLES

The examples below are not limiting and are merely representative ofvarious aspects and features of the present invention.

Example 1 Targeting Vectors

Preferred targeting vectors include four major elements. A promoter,preferably the protamine gene promoter, is linked to, and drives, theexpression of a gene, preferably the hamster BiP protein, to disruptsperm development. Both wild-type and mutant hamster BiP genes may beused to prepare vectors. The third element of the vectors is a Y or Xchromosome specific DNA sequence which is linked to the promoter/geneelements. The Y or X chromosome specific sequence is to be used ashomologous arms for targeting the vector to the Y or X chromosome,respectively. The fourth element of the vectors is a selection marker,such as the neomycin-resistance gene, neo (Southern, P. J. & Berg, P.(1982) J Mol Appl Genet 1: 327-341).

Preferred elements of the vectors which may be obtained and incorporatedinto the targeting vectors include novel sequence of both the bovine(Lee et al (1987) Biol Chem Hoppe Seyler 368: 131-135; Krawetz et al.,(1988) J Biol Chem 263: 321-326) and porcine (Maier et al., (1988)Nucleic Acids Res 16: 11826) protamine promoters. In addition, apreferred toxic gene, a dominant negative mutant of hamster BiP protein,plus wild-type hamster BiP protein, to disrupt proper protein folding inX- or Y-bearing sperm have been disclosed (Hendershot et al., (1996)Proc Natl Acad Sci U S A 93: 5269-5274; Morris et al., (1997) J BiolChem 272: 4327-4334). Suitable bovine and porcine Y chromosome specificsequences 3′ of the SRY gene (Hacker et al., (1995) Development 121:1603-1614) to be used as homologous arms for gene targeting have alsobeen disclosed.

As described below, complete insertion vectors containing the bovineprotamine promoter, mutant or wild-type BiP cDNA, the neomycin-resistentmarker neo, and a homologous arm with bovine Y chromosome specificsequence have been constructed. The backbone for the insertion vectorwas pGT-N29 (New England Biolabs #N3729S). Preferred insertion sites areXho I and/or Bsi WI in the vector. A diagram of the constructs is shownbelow.

Promoter Sequences

In 1988, Krawetz et al. published a bovine protamine 1 gene cDNAsequences with 597 bp of 5′ flanking region. Krawetz et al., (1988) JBiol Chem 263: 321-326. In order to obtain a more complete promotersequence, PCR of bovine genomic DNA was performed using forward (nt615-640) and reverse (nt 1003-1028) primers from the publishedsequences. A fragment of genomic DNA containing the cDNA and the intronof protamine 1 was obtained. The fragment was used as a probe toisolated a cosmid clone from bovine genomic library (Genome SystemBovine Cosmid Library, clone address 180P13).

According to the data obtained, it was determined that nucleotide 1 to207 of the published sequences of bovine protamine 1 are actuallyprotamine 2 sequences which were mistakenly assigned to the protamine 1sequence. Thus, the actual sequence of protamine 1 begins fromnucleotide 208 of the published sequence, and contains only 390 bp ofthe 5′ flanking region. In addition, a ˜1 kb sequence which is locatedfurther upstream of the protamine 1 gene was obtained. TABLE 1 Publishedbovine protamine 1 gene sequence (5′ to 3′) (SEQ ID NO: 1). 1 TCGAAACCAGGGGACAAAAC CTCTGAAGAT GAGGGCCAGC CTCCTTGTCT GGATCCAAGC 61 CCTCACACCCTGCCCCTCCC CCAGCTCCTC GGGGTTCCTG AAGCTTCCCT GCTGCCTTTG 121 CAGCCACTGCTGTGGCCTCT CGGGGGGCTG GGATGGGGGC TTATCTGTCC ACAGGGTTAT 181 CTTATGCTCACTCTGTGCCA G

CTC CTTTACAGAG GAGGAGGCAT GGAGACTTGG 241 ACGTCATAGC TGGGTTCGGGCTGCTCATGG GGTCTTGGAC CAGCTTGGCA GGAACTGTCA 301 TGACTCCTCT ACCTCCCCCCCCTCCCCACT GCATGATGTG ATGTGGTCAA ATTTATATGC 361 ATTAATGACC TGGGGGGTCATTAATTAATG TGGAGGGGCC CCACCCCCCC CCACATCACA 421 GCCCCACCCC TGCACATCACAGCCCCGCCC TCCCTCACCA AGCACCTCCC ACATGCCCAT 481 ATATGGGCAT GATTTGGGCAGCTCTGACCC TGGTCTGTGA GGTCTGGGTC TCTGTGACCT 541 CACAATGACC AGGGCCCTGCCCGGGTCTAT ATAAGAGGCC AGGAAGTCGG CCCCTGTC*AC 601 AGCCCACAAA TTCCACCTGCTCACAGGTTG GCTGGCTCAA CCAAGGCGGT ATCCCCTGCT 661 CTGAGCATCC AGGCCGAATCCACCCAGCAC C ATG GCCAGA TACCGATGCT GCCTCACCCA 721 TAGCGGGAGC AGATGCCGCCGCCGCCGCCG AAGAAGATGT CGCAGACGAA GGAGGCGCTT 781 TGGTCGGAGG CGCAGGAGGA

 

841

 

 

 

 

901 GCAGTGTGCT GCCGTCGCTA CACCGTCATA AGGTGTACAA GACAGTAACC ACACAGTAGC961 AAGACCACCG CACTCCTGCC TGAAAGGTCA CCAGCCTTCA AGACCCTCTT GCCACATCTT1021 GAACATGCCA CCATTTCAAT GACATGAACA GGAGCCTGCT AACGAACAAT GCCACCTGTC1081 AATAAATGTT GAAAGACATC ATTCCACTCT TTGACTCTTT GCTTTGAGGG ACTCTAGGCG1141 GGGTGGGGGG GGGGGGGAAG GAGGGGGTTG GGGATGCTGG ATCTTGTTCC AAACTCAACT1201 ACTCCCGAGT CACAAACCAA ACCTGCCTCC CAGCCCCTAG TCCTTTACAG ACCCCTTTCC1261 AGCGGGGACG GGAGCTGTGC TGGTTGATGA ACACATCCCT CCCCAGTTCT GTGCTCAGTG1321 GCTTTCTACT GACAGCTCGA

The EcoRI site at nt 202-207 is italicized and underlined. The starindicates the transcription start site and the atg start codon isunderlined. The italicized and bold sequence is the intron 1 region ofprotamine 1.

The sequence obtained in the present invention are provided below inTable 2. As obtained and presented, this sequence is reversed, and iscomplementary to the sequence shown in Table 1. The first 48 nt matchwith nt 249-202 of the published sequences (thus the first threenucleotides (CTA) in this sequence are complementary to the threenucleotides beginning at nt 247 of Table l(reading backward, GAT)).TABLE 2 Sequence of the 5′ flanking region of bovine protamine 1 (3′ to5′) (SEQ ID NO: 2) CTATGACGTCCAAGTCTCCATGCCTCCTCCTCTGTAAAGGAGGAATTC

TGCAAGAAATCAAAGGGAGGCCGAGGGGGACGGAGCANGAGAGTGCGGGGGAAGGGTGGGCACAACAGATAAGGAAGGTAGCAATTAGAATTTGAAATCGTTACTCATAGCAGGAAACCAAAATAAGTGTCTTTGGCATGTGNNGGNGGTTTAGTCACCAAGTTGTGTCCAACTTCTTGCAACCCCATGGACTGTAGCCCGCCAGCTCCNTCTGTCCATGGGATTCTCCAGGCAAGAATACTGGAATGGGTTGCTATTTCCTTCTCCTGGGGATCTTCCCAACCT -5′

A preferred promoter sequence used in the present invention is shownbelow in Table 3. This promoter sequence is shown in the sameorientation as that of Table 1, and is thus the reverse complement ofthe sequence in Table 2. The sequences contain nt 202-nt 690 (before theatg start codon) of the published bovine protamine 1 sequence (shown initalics) and 852 bp of sequence obtained in the present invention (shownin bold, also shown in FIG. 2 in italics, including the underlinedregion). TABLE 3 Preferred bovine promoter for use in the bovinetargeting construct (5′ to 3′) (SEQ ID NO: 3)5′CCTTCCAAGCAACTTTCAAGCCTAAGACTTTTTTTTTTTTTTAACCCAATTGAGGGTAAAAATTCACATCGTGGTAAAATCCAGCATACTAAAAAAGTGAAAATTCAGTGTCAATGAAAACATTCACCATGTTGGGTAACTGTCATCACTATCTGGGTTCAAAACATATTTATTCACACCAAAAGGAGCCCCTGCACCGCTTATGGAGCAGCCGCTCCCATTTCACCCGCTTCTGGGCAACCACCAACCCATTCCTGCGCCGGTGGCCTTCCCTCAGTGGCTGTTTCACTACTGGAATCACACACGTGCTGCCTCAGGACTTGGCAGCAGCTCATCCCTCTTCCCAGATGCTCTTCCCTCCTCCCACTCCAATCATCTTCCTTAGGAGGCCTTCCTGGGGCCTCTCCTGAGGTCAAGTCCTCCAGCATAACTTTCTCATAAAAGCCCAGAGTTTCTCTCTGAGGCATTTTTTTCACAAGTGCTCTGCATGGTAATTTCATTGGGTTTCCCTGGTGGCTCAGACGGTAAAGAATCTGCCTGTGATGCAAGAGACCCGGGTTCGATCTCTGGGCTGGGAAGATCCCCTGGAGGAAGCATGGGCAACCTACTCCAATATTCTCGGCTGGAGCACTCCATGGACGGAGGAGTCTGGTGGGCTACATATAGTCTGTGGGGTTACAGTTCATGTTCTCTACATTAAGAATGTAAAGTTTATTAGGCAGGAACATGTCTGACTTGTTTACCAGTATTTGCCAGGGCCTCGCCAGAACCTGGCATACACTGTAAACTGACTGAGTGGATGAGCTAGTGAGTGTCCTTACAAAGAAAAAGTCACCTCATTCCTCTTTACAG AATTCCTCCTTTACAGAGGAGGAGGCATGGAGACTTGGGCCGTCATAGCTGGGTTCGGGCTGCTCATGGGGTCTTGGACCAGCTTGGCAGAACTGTCATGACTTCTCTACCTCCCCCCCTCCCCACTGCATGATGTGATGTGGTCAAATTTATATGCATTAATGACCTGGGGGGTCATTAATTAATGTGGAGGGGCCCCACCCCCCCCCACATCACAGCCCCACCCTGCACATCACAGCCCCGCCCTCCCTCACCAAGCACCTCCCACATGCCCATATATGGGCATGATTTGGGCAGCTCTGACCCTGGTCTGTGAGGTCTGGGTCTCTGTGACCTCACAATGACCAGGGCCCTGCCCGGGTCTATATAAGAGGCCAGGAAGTCGGCCCCTGTCACAGCCCACAAATTCCACCTGCTCACAGGTTGGCTGGCTCAACCAAGGCGGTATCCCCTGCTCTGAGCATCCAGGCCGAATCCACCCAGCACC 3′

A Clontech Genomic Walking kit was used to isolate a promoter sequencefrom the porcine protamine gene. The two walking primers used based onknown sequences were: (SEQ ID NO: 4) PP1W1:5′ GACTTCCTAAAGGATGAGTCAGAGTTGGAGG 3′ (SEQ ID NO: 5) PP1W2:5′ GGAACAGCAGGTGCTAAGTTCTGAGGCAG 3′

A ˜1.0 kb fragment was amplified and sequenced, and the sequenceobtained is shown in Table 4. The underlined sequence matches nt 1 to nt47 of the published sequence. A preferred sequence for use in a porcinetargeting construct contains nt 1- nt 694 of the published porcineprotamine sequence and 954 bp of sequence obtained in the presentinvention (bold italics), as shown in Table 5. TABLE 4 Porcine protamine1 promoter 5′ flanking sequence (5′ to 3′) (SEQ ID NO: 6) 5′GAGAGCTTCTAGAGAAGAGTCTCAAGAACCATACAAA

TTTCATGAAAATGGAATCACACACTATGTGCTGCCTCA 3′

TABLE 5 Preferred porcine promoter sequence (5′ to 3′) (SEQ ID NO: 7) 5′

TTTCATGAAAATGGAATCACACACTATGTGCTGCCTCAGAACTTAGCACCTGCTGTTCCTTCTTCCCAGATGCTGTTCCCTCCTCCAACTCTGACTCATCCTTTAGGAAGTCCCTTCACCAGCATTTCCTCAGGAGGCTTTCCTATGGCATCCCCTGAGGTCAAGACCCGCCTCCCCAACATACATCCTCATAAAATCTCTGAAGGTTCTCTCTCTCAGCAATTTTCATGATTATAATTACTCTGTGTGGTCATTTCATTCATGTCTCCTGGAGTTAGATTATAAAGTTGACTAGGCAGGAACATGTCTGCCTTGTTTATCACTGTATGCAGGGCTTGCCAGAATCTGGCAAACATAGGGGCTCAATAATAATTTGTAAACTATCCGAGTGAATGAGTGAGTGTCCTTACAGAGGTCACCTCGTGTCCCTCTGCGGATGCATCACGGCCCCGCCCTCCCTCACAAGGCCCTCCCACATGCCCATATATGGACACGATGCAGGCCGACTCTGGCCCTGGTCTGTGAGGCCTAGGCCTCTGCGACCTCACAATGACCAGGGCCCTCCCCGCGTCTATAAGAGGCCCAGCAGTCAGCCCCTGGCACACAGCCTCCAAAGTTCCACCTGCTCACAGGTTGGCTGGCTCAACCAAGGCGGTATCCCGTTCTAA 3′

The skilled artisan will understand that one or more nucleotides may bedeleted, substituted, and/or added to a promoter sequence, while stillproviding a functional promoter. Preferred promoter sequences are thosein which no more than about 2% of the nucleotides differ by deletion,substitution, and/or addition from the sequences disclosed herein; morepreferably no more than about 1% of the nucleotides differ by deletion,substitution, and/or addition from the sequences disclosed herein; evenmore preferably no more than about 0.5% of the nucleotides differ bydeletion, substitution, and/or addition from the sequences disclosedherein; and most preferably no more than about 0.1% of the nucleotidesdiffer by deletion, substitution, and/or addition from the sequencesdisclosed herein. The term “about” in this context refers to ±10% of agiven percentage (e.g., about 1% refers to from 0.9% to 1.1%).

Expressed Transgene Sequences

A preferred gene for use in disrupting sperm function is the dominantnegative mutant of hamster BiP protein disclosed in Hendershot et al.,(1996) Proc Natl Acad Sci USA 93: 5269-5274. This dominant negativemutant has been shown to cause improper protein folding and abnormalexpansion of ER in monkey cells (COS cells). Expansion of ER may affectthe compaction of sperm head during spermatogenesis and improper foldingof sperm surface proteins would disrupt the function and motility ofsperm. Since BiP is a native ER protein, it is less likely to diffusethrough the cytoplasmic bridges connecting the developing spermatids. Ifthe mutant BiP is expressed in X- or Y-bearing sperm by targeting theBiP cDNA to X or Y chromosome, it may disrupt the function of the spermpopulation that expresses it. The sequence of wild type hamster BiP isshown below in Table 6. The dominant negative mutant of BiP is identicalto the wild type with the exception of a change in the codon at nt 259from ACC (coding for threonine at amino acid 37) to GGC (coding forglycine). The preferred segment of the gene that was used in the presentinvention is bounded by the nucleotides indicated in bold underline; thestart and stop codons of the coding segment of the gene are indicated initalic underline.

Table 6: Hamster BiP cDNA sequence (5′ to 3′) (SEQ ID NO: 8) TABLE 6Hamster BiP cDNA sequence (5′ to 3′) (SEQ ID NO: 8)GACACTGGCCAAGACAACAGTGACCGGAGGACCTCGCTTTGCGGCTCCGAGAGATCGGAACGCCGCCGCGCTCCGGGACTACAGCCTGTTGCTGGACTTCGAGA C TGCAGACGGACCGACCGCTGAGCACTGGCCCACAGCGCCGGCAAG ATG AAGTTCCCTATGGTGGCGGCGGCGCTGCTGCTGCTCTGCGCGGTGCGGGCCGAGGAGGAGGACAAGAAGGAGGATGTGGGCACGGTGGTCGGCATCGACCTGGGGACCACCTATTCCTGCGTTGGTGTGTTCAAGAACGGCCGCGTGGAGATCATAGCCAACGATCAGGGCAACCGCATCACGCCGTCGTATGTGGCCTTCACTCCTGAAGGCGAGCGTCTGATTGGCGATGCGGCCAAGAACCAGCTCACCTCCAATCCCGAGAACACGGTCTTCGACGCCAAGCGCCTCATCGGACGCACTTGGAATGACCCTTCAGTGCAGCAGGACATCAAGTTCTTGCCTTTCAAGGTGGTTGAAAAGAAAACTAAACCATACATTCAAGTTGATATTGGAGGTGGGCAAACCAAAACATTTGCCCCAGAAGAAATTTCTGCCATGGTTCTCACTAAAATGAAAGAAACTGCTGAAGCATATTTGGGAAAGAAGGTTACCCATGCAGTTGTTACTGTGCCGGCTTACTTCAATGATGCCCAGCGCCAAGCAACCAAAGATGCTGGCACCATTGCTGGACTGAATGTCATGCGGATCATCAATGAGCCCACAGCAGCTGCTATTGCGTATGGCCTGGATAAGAGAGAGGGCGAGAAGAACATCCTCGTTTTTGACCTGGGCGGTGGAACCTTCGATGTGTCTCTTCTGACCATTGACAATGGTGTCTTTGAAGTGGTGGCCACGAATGGAGACACTCATCTCGGTGGGGAAGACTTTGATCAGCGGGTTATGGAACACTTCATCAAGCTGTACAAAAAGAAAACTGGGAAAGACGTTAGAAAAGACAACAGAGCTGTGCAGAAACTTCGTCGTGAGGTGGAAAAGGCTAAGCGAGCCCTGTCTTCTCAGCATCAAGCAAGAATTGAGATAGAGTCCTTCTTTGAAGGAGAAGACTTCTCTGAGACCCTGACTCGGGCCAAATTTGAAGAGTTGAACATGGACCTGTTCCGATCTACCATGAAGCCAGTCCAGAAAGTGTTGGAAGACTCTGATCTGAAGAAATCAGACATTGATGAAATTGTTCTTGTCGGTGGGTCTACTCGGATTCCCAAGATTCAGCAGCTGGTGAAAGAGTTCTTCAATGGCAAGGAGCCATCCCGTGGCATAAACCCAGATGAGGCTGTAGCATACGGTGCTGCTGTCCAGGCTGGTGTCCTCTCTGGTGATCAAGATACAGGTGATCTGGTACTGCTTGATGTATGTCCTCTTACACTTGGTATTGAAACAGTGGGAGGTGTCATGACCAAACTGATTCCAAGGAACACTGTGGTACCCACCAAGAAGTCTCAGATCTTTTCCACAGCTTCTGATAATCAGCCAACTGTAACAATCAAGGTCTATGAAGGTGAACGACCCCTAACAAAAGACAACCATCTTCTGGGTACATTTGATCTGACTGGAATTCCTCCTGCTCCTCGTGGGGTACCCCAGATTGAAGTCACCTTTGAGATAGATGTTAATGGTATTCTTCGAGTGACAGCTGAAGACAAAGGTACAGGGAACAAAAACAAAATCACAATTACCAATGACCAAAATCGCCTGACACCTGAAGAAATTGAAAGGATGGTTAATGATGCAGAGAAGTTTGCTGAGGAAGACAAAAAGCTCAAAGAGCGCATTGATACCAGGAACGAGTTGGAAAGCTATGCTTACTCTCTCAAGAACCAGATTGGAGATAAAGAAAAGCTGGGCGGTAAACTTTCCTCTGAAGATAAAGAAACCATGGAGAAAGCTGTAGAGGAAAAGATTGAATGGCTGGAAAGCCACCAGGATGCAGACATTGAAGACTTTAAAGCTAAAAAGAAGGAACTAGAGGAAATTGTTCAGCCTATTATTAGCAAACTCTATGGAAGTGCAGGCCCTCCCCCAACTGGTGAAGAGGATACATCAGAAAAAGATGAGTTG TAG GTGTACTGATCTGCTAGGGCTGTAATATTGTAAATATTGGACTCAGGAACTTTCGTTAGGAGAAAATTGAGAGAACTTAAGTCTCGAATGTAATTGGAATCTTCACCTCAGAGTGGAGTTGAAAATGCTATAGCCCAAGTGGCTGTTTACTGCTTTTCATTAGCAGTTGCTCACATGTCTTGGGGTTGGGGAAAGGAGGAATTGG CAATTTTTAAAATT

Y Chromosome Targeting Sequences

Targeting a specific chromosome site is usually accomplished byinsertion of a construct (containing a gene of interest and preferablycontaining a selectable marker, often neomycin resistance) into a hostgenome, causing disruption of splicing, promoter function, or readingframe, with or without deletion of the targeted gene. Incorporation ofthe construct into the genome depends upon insertion into, orreplacement of, the endogenous gene by homologous recombination throughone or more arms of the construct into one allele of genomic DNA. Asstarting material, a genomic clone of reasonable length must be obtainedfrom a host genome. For adequate frequencies of homologousrecombination, typically at least about 1 kB of uninterrupted sequenceis used as a homologous arm, preferably at least about 2 kB, morepreferably at least about 4 kB, even more preferably at least about 6kB, and even more preferably about 7 kB. When more than one homologousarm is used, the arms need not be equal in length (e.g., one arm maycontain about 4 kB of sequence, the other about 2 kB). The term “about”as used in this context refers to ±1-% of a given dimension.

To target the transgenic construct to the bovine Y chromosome, a bovineSRY sequence was used as a probe to screen a bovine BAC library toidentify sufficient sequence to act as a homologous arm. The primersused for library screening were: (SEQ ID NO: 9) SRYF3: 5′ GCA CCT GTGAGA CCC AAG GTT TCA TCT C 3′ (SEQ ID NO: 10) SRYR1: 5′ CAC CTC ATC AGATTA ATC AGA CAG G 3′

In the present invention, a BAC clone containing the bovine SRY gene wasisolated, and the genome sequenced towards the 3′ end of the gene. About11 kB of sequence downstream of SRY on Y chromosome was identified, asshown in Table 7. A 6.6 kB segment of the sequence was used as thehomologous arm in the insertion vector, as shown in Table 8. TABLE 7 An11 kb sequence 3′ of the bovine SRY gene (5′ to 3′) (SEQ ID NO: 11)TTTGAGGCGATTATAACATCCATCCAGTATTTAATTAGCACCTGTGAGACCCAAGGTTTCATCTCTTTTCTGAAAATTTTCTTTTAATCACTGGCAATAAATACACTTGTTTCCATTTTCACTTAAGTTTTGCATTCTTGGAGGGAGAAAACAAAAAATAATAGTGCCTTCATATCAAGAATATAAATTATTCAGATTATGTGGCATGGGGGATGGGATAAACAAGATCCTGTCTGATTAATCTGATGAGGTGTCAGTGAAAATGTAAATCAAAGGTGTTCTAAAAATTTGCAAATAGGCTAAAGTAGAAAAATTGGCTACGCTTGCAAAGGAAGCATCCCTTTTTTGGAATGAAAATAAGTCCATGGTGAAACTGTATGATATGATATGATATATATTACATATTAAAGTCAACTTTCCAATACATATGTTCCAAACTTTTGAAAAACAGTACTTCAAATAATAAATCTCAAAACCCAAAACAATATGTAGTGAAATGTGGAGTTTTTGAAAGAAAGTTCAGGGAAAAAAGGGAGGTAACTTCACAGACTGTTTTATTCCAGGAAAAATATTGTTTAATCAGACATTTATGCATTCCAAATGGTAATTATGTGCTATGATAACCTTCTAAACAAATACCTCCAGGTTGTATTTTAAAGTATTTCTATATTCTTTCTATTTATATGTATTGGTGTTTAATATTTTAAGCCTCTGTTTCCACATGTTCATAAATAAACTGTACTTTAACTTTTGTCAAAATAGGTATGCTTTCCTTTTCTTTAACTTCAAATAAAGGAAAACATATAATACTATGTTTAATTTACTTTGCTTTAAATAACATCAGTGACACTGAGTTTGTTTTGGAAATTACTATGTACACTTCATCCTGTATACTATAGATGTATAACTGTGTTTCAGGGAGGAAGCTGGATTCTGATTCCATGTTGGAAATTGTTTCTTTACTTACCTTTTATTTTTATAACCATTCTAAATTGCTTGCCTGGGGGACTCTGCCCCTTTTGCTTGACTGTAAACTAAAGTGTCTTTGTTTTGCTCAAAAAGAAATAGTTTGTCTCTGTTTACCTGTGAATAGAAGAGATTAACACACTCCTGAAGACTGGACATTCCCTTGAAGAGGTTTTATAAGACTGAAGATCCTTCTATTTTATTTTCCCCCTGCCTTTCTCTCTATTCTGGCTTTTGACTTGAGTTCCTCATGCTTCTTTTTCTCTGATCTAAACAGAACCTGGTATCCAGACCCTAATAAGATAATTATTTTGAGGCACTAGCCTGCCTCTTCTCAGTCTGCCTGCTCTGTGATTAAAGTCTTCTCATTGTGTCAACAACTTGTCTCTTGGATTCATTGGCCTGTCATGAGGTGACCAGAGTGAGCTTGGACTCGGTAACAATTGCACTTTTGGGCTTTAATTATTATGAGTAATGATGTCCTTTGTACATTTGTATACCCGTCTGTGGTAGAACATTTACAAACATTTCTCCTGAATATATGTCTAGGAAAGAAATGAATGGTTTTGTGTATTTTTAGCAAGCTCTTTTTTTTTCTTTTTTTGTAAATTTTGATGAATATTCTTGTATTTTCTAATGAATATCTGCTATATTTTAAAATGTGCCAACTTTTAAAAATATTCATTGGTATGAACTAATACCATGAATTCCAGATGTAATTGGATATGACTCCTTTCTCTACCATTTATCAGGGCTGACATTGATGGATTTGTTTTGGTCCTCAGTTTGTTTACCTTTAATCAGCAGTAGGAATAATAATAAATTAAAATAAAAAACAGCAAAGCAGAAAAATAAATCCTCATTGGGATGCTGGGAAGACTATGTAACTTTAAGGTGTATAATGAATCAATGAGCAAAAATATATAAAGCATTACAATTAAAAGTCAACATTAATTAATGCTAACATTAACTAATTAGTATGTTAACTAACACTAACATACTAACACTAACTAGTATGGATGATAATTATACAAAATAAAAATGACAAAAGCTACCTACTAGGATCTGTGAAAACTAAATAAATGAAGGTCATATTCTGTGAGTGAAAGTATGCATAGTACTCAGAACAGAGATAAGCACTGGTAACTAACAACTGTTGACTGAAAAATGACAAGAGTTGTGAATTAAGTTTTCTTTGGGGCAAAATGAGGACTGCAGCCCAGGAGGCAGCATCAGATAGCTCTAAGAGACTACTCCAAAGTGGCAGTGGGGGAAAGTCAATATATAAGGTTTTGGTGAAGGGGGAGTTCAAAACCATGAACTGCTCATTTTACAAGAGGTTTTTTTGTTAGTCATGAATATCTGATGTCACCATGAGGGGATTTAGTGCTTACTCTATATATGAGGAGATGCAAGTATTGAGATCATACAATGTAATCCTAAAGCATCCATCTATCTAAAGACCTGTCTCGAAGACAGCCTCACCCTGAACTCCCTCAGGGTTGTTGAAGGTCAACAGCATGAGGTTCAATCACCATAGAGGCAGATGGCAAACACCTTTGTTGTTCAGTTGTCGGCCAATGCTCTTGATAGATGCCAATTTGTAGTTGACACAACTAATTAACAGAGAAGGCAATGGCAACATACTCCAGTACCTCTTGCCTGGAAAATCCCATTGGATGGAAGAGCCTGGTAAGCTGCAGTTCTATAGGTCGCAAAGAGTCGGACACCACTAGTGACTTACTCTGACTTTTCAGTTTCAATGCATTGGAGAGAAATGGCACCTACCCCCAATGTTTCTTGGCCTTGGAGAATCCCAGGGATGGCAGAGCCTGGTGGGCTGCTGTCTGTGGGGTCACAAAGAGTCGGACACAACTAAAGCGACTTACCAGCAGCAGCAGCAACAAATTAAAATAATGATAACAAATAACAGTGACAACCTACCTCAATTGGATGCAGAAAGGACAAAATTACTTAAAGATGCATAATAATTCAAGGATTTAAAGTATTTAAAGTACTTAGGGTAGATGTAGGCACTGGTAAAGAAATAAATAGTTAGAATAATTAAAAAAATAAACAGAAGGACAAAAACAAAAACAAAACCTGTTCTGCTTCATTGAGATGCTGTGAAGACTGAAGAAACTATGATGCATACAGATTTAATGAATATTTAATATATTCAGAGGTTAACATTATTAAAGTGCTAAATAGCAATAATGATAATGATGGTAACAATGATAATGATATTATAATAATAAAACCCCTCACTGGAATATTATGAGACTAAATAGGTAAAGGTATGTAAGGTTCAAGAAATAAATATACAATGTTCTTACAGTAAAAGATAACATTAGGAAAGAAGTAATTAATAATAATTACAAATATTAATCATGATAATAAATAACAGCAAATCTTTCCTTCAGGGGATTCTGTGAAGACTAAATATGAAAGTATTTAGATTCAAAGAGTAGATGTATATAATGTACTAAAAATGGAGTTGTTTTATGATGTGTAGCTATAGCAATAATGAAAGCAACAATGACATCATTTGATATGCCTGTGAAGACTGAATAATTTCAAGTGAGCAGAGTTCAAGGAGCACAATGTACTGCAAATTAAGGTCAGTTTTAATAGAGAAAAAATCAATACTAATAATAATTCCAATAGCAATAATAGTACAAATATAGCAATGATGGATACTTAACTAGGATGCTATGAACACTAAGGAAATTAAGACTTAAAGGATTTGATGAGAAAGTGTATCTAAAGTACTAAGAGAAGAAAGTCAACATGAGTAAAATCTAAGTAGTAATAATAATAATTATGAGGATGATGATGATAAAGTAGAAATAAAACCTACTTCAGGGATGCTGTGAAGACTAAGTGAAGGTGTAGGATTCAAGAAATAAGTATTTTGAAATACTTGGAACACCGATAGATATTAGTAAAACACTAATTAATAACACCACCAACATGAATAATAATAAATAATAAAAATGAAACACATCATTGGGATACTATGGCAGTTTTTTAACTAAGTTATGGTATATAGGGGCTGAATGAGTAAATGCATAAAGAAGTACTTAGAAAAGAAGGATTGGAGACAAGATGGCAGACATTTGTCTGAACGTGAAAGAACACTGAATGAACACTGAAAGATGAACTCCCCCAAGTTGGGAGTGACCAATTGCTACTGGAGAAGAGTGGAGAAGAGCTCAGATGAATGAAGAGGCTGAGTCAAAGCAAAACAACTCCAATGGTGTAAAGAAAAATATTTCATAGGAATCTGGAATGTTAGGTCCATGAATCAAGATGTTGGGAAGGCTGGGAGGAGGGAAAGAGAGATTCCATCTTGAAGACTGTCAGTTATCTTAAGGCACGATGAAAACTGGGCCTGAACCCTGTTAACTATTGTCAAACTAAAGTCAGGAAACTCCATCCTCACAGATGGCAAAGATTGGAAGTAAAGGTCAGATTGTGTTAGACTAACGATAGTGCCTGAACGTAAAGGTCAGATTGTGTTAGACTAATGAGAGTGCCTGAACCTGCATGTTGTAGTTGTTAATTCTTCCACACCTGCATATTGTAAAACAAATTACTAATGTGTAACCAGTTTGAGTGAACTCTGGGAGTTGGTGATGGACAGGGAGGCCTGGTGTGCTGTGATTAATGGTGTTGCAAAGAGTCGGACACAACTTAACGACTGAACTGAACTGAACTGAACTGAATGTGTAACCATTCATGTAGTGGAGGGTATAAAACTGAGTCCTCCAAAATCATCAAGGTCCTTGTCAGAACCGATTCCCTTGGGCCTGTTATGTGTAATAAAACTGTTCACTATACTGAGTGTCCTCCAAGGATTGTTCTACAACTCTGGATTCTACAAAATACCTGGTGTGTTGGCTGTGAAATCCTCAGAGAGAGAGGCACATTGAGCCTCCACCTGAGGCTTTCACTGGGATGAAAGCTTCTGTGAGGGGATGGCACCTCCTCTCTTAGATCACCTCTTGTTTTATTGACTCATCTTTCTAAGCAGACTTCACAAGACTGTGGATTACAGAGGGAAACACTCAAGTAGGTCCCACTGTAATAGTGGAAGAAGGGGCCTGATCAACTTATTGGGGCTGGATGAACCTGTGGTGACTGTGTCCTTGTAGGCTCAGTGGGGAATTGTTTACTGAAGTAAGTAAAACACTGTTAACAGAATCTGTGCCAATTGTTTGTCAATGTCTTACTGGTTTCCAAGCGACTGTCCAATTTGTGCAACACCCTCCCATTCTCCTAGGCATTCAGGGACTTCCTGAATGTTGTTTATGACCAGACTGAAACTGAGCTCTGGGTGCACATTTTGTCTGCACTGACCCATAATAAGACAGACTGGGATCTTGCCAGGATCAGACTTATGCCAAAGAGAAGTATATTGAAAGCCTTAATAGATTGGATTTGGATGACTGCTAACAGAAAAACATGAGAGATAATACCATGGAAAGACTTGGTAGGTAAGACCCTTTTCACTTTGATCATAGTTGAGGTACAGTGGCCCTTGTCCTTCTTTGAGTGGAGACTTCAGTCAGGGCTGGGGTACAAGACCCTAGCAATGAGCGATGAAATAGAAGTTGGACTTGCTGTAAGTGATAGAAGGAAAGTAAAAAGTAGGAAAGGTAGCAGAGAATTCAAACCAACCTTTCCTGAATGGATGATTAAAATTTCAAAAGGGTTTTGGAGGAAACAAACAAGCAAAAAGTGAACCCCAGTCAAGCTTAGAACATTCTGTGAGCTAGACTGGCCATTCTTTGGGGTAGGATGGCTCTCAGAAGAACCCTATGATTGGACAAAGTTTGTACTCTGTGGTTACTGAACAGCCAGGACACCCAGACCAGTTCCTGTACTTATGGCTACTAACTGCACAAAATCCACATCGTTAGGCCCAGGTATGTTATCTTGGAAAAGGAGAGAGCAAGATTTTGCTGGTGAAGCAAAATCCAAGAAAGAAAAATTAAAGGAAATATGATCTGGAGGAGTAGGAACCTCCCACCATGCCCTCTCATTCTGGAGAACATGAAGGGGATCCCCAGAAGAAGAGGAAGGGACATTCCTCCTCTGCGTCCCCCAGTGGAGGAGGTTCTCTCTTCCCTCCTATTGTACCAAAAATCGTGACAGGAGCTACTGCATCAACATTATACCCAACCCTCCCCAGTTTAGAAGAGGAGAGAAAGGGAGAGTTAAGTGTTCATAGGCAGCTGAGGTTCTACAAAGGAACCTCAAGAGAGAGAGGTTTTGCAAATACCTTTTACGGAGGTTCAAGCAGTATCACAGGTGGGGCCAGATGGGCATATCCATCCTGGCCACACTGTTCTTTTCTATCAGCCATTCTTTACCACTGGTCTTTTGAACTGGCAAAGGCATACCCCTCCCTATTCTAAGAAACCATATGGTCAATTCATCGGACAAAGATTATTTTCAGGATCACCAACCTGTGTGGGATGACATAGCCCAGCTTCTCCTCACCCTCGTCAGTACAGAAGAAAGACACCGGGTCCTCCCAGAAGTATGTAAATGGCTTCACTGGTAAAATTTCAGGTGGGAATTTTTTCCTCATTTCTGTGGTGATACCAAAGGGAAGATGAGTGGAAGCTCTAACAGTCATCTGGAGGGCTACAGACCCTAGAACCCCTAACAAAGCTGTTTCCTGAAGTATAGGCTAAAAGCAATCCCCTGGACTTGCTGAAAACCACCCTCTGGTGATAATAGAGCTAAAGGTGGGAGCCTAGCCCATAAGGAAAAAACAATATCCTATACCACTGGCTGCCAGGGAGGGAATCAAGCTTTCACATTGATAGGCTGAAGGGCACAGGCATACTGGTGGAATGCCAATCACCATGGAACACCCCTTTCCTCCCAGTAAAGAAGGACAGGGAAAAAGATTATTGGGAAGCTCAGGGCATGAGATCCCAGCGATGGGTCAGAAAACCCAGCTGACTTGGATTCAGGCTACCACAAGGATTCAAAATTTCCTTACAATATTTGGGAGATGCAGACAAAAGAACAAAGCTAGTGGACGTGATGGAATTCCAGTTGAGCTATTTCAAATCCTGAAAGATGATGCTCTGAAAGTGAGGCACTCAATATGCCAGCAAATTTGGAAAACTCAGCAGTGGCCACAGGACTGGAAAAGGTCAGTTTTCATTCCAATCCCAAAGAAAGGCAATGCCAAAGAATGCTCAAACTACCGCACAATTACACTCATCTCACACGCTAGTAAAGTAATGCTCAAAATTCTCCAAGCCAGGCTTCAGCAATACGTGAACTGTGAATTTCCTGATGTTGAAGCTGGTTTTAGAAAAAGCAGAGGAACCAGAGATCAAATTGCCAACATCTGCTGGATCATGGAAAAAGCAAGAGAGTTCTAGAAAAATATTTATTTCTGCTTTATTGTCTATGGAAAAGCCATTGACTGTGTGGATCACAGTACACTGTGGAAAATTCTGAAACAGATGGGAATACCAGACCACTTGACCAGCCTCTTGAGAACTCTGTATGCAGGTCAAGAAGTAACAGTTAGAACTGGACATGGAACAATAGACTGGTTCCAAATAGGAAAAGAAGTACACCAAGGCGGCATATTGTCACCCTGCTTATTTACCGTGCAGAGTACATGCAGAGTACATCATGAGAAATGCTGGACTGGAAGAAACACAAGCTGGAATCAAGATTGCAGGGAGAAATATCAATAACCTCTGATATGCAGATGACACCACCCTTATGGCAGAAAGTGAAGAGGAACTAAAAAGCCGCTTAAAGAAAGTGAAAGTGGAGAGTGAAAAAGTTGGCTTAAAGCTCAACATTCAGAAAACGAAGATCATGGCATCTGGTCCCATCGCTTCATGGGAAAAAGATGGGAAACAGTGTCAGACTTTATTTTGTTGGGCTCCAAAATCACTGCAGATGGTGAGTGCTGCCATGAAATTAAAAGCACTTACTCCCTGGAAGGAAAGTTATGACCAGTTTAGATAGCATATTCAAAACAGAAACATTACTTTGCCAACAAAGGTCCGTCTAGTCAAGGCTATGGTTTTTCCTGTGGTCATATTTGGATGTGAGAGTTGGACTGTGAAAAAGACTGAGCGCTGAAGAATTGATGCTTTTGAACTGTGGTGTTGGAGAAGACTCTTGAGAGTCCCTTGGACTGCAAAGAGATCCAACTAGTCCATTCTGAAGGAGATCAGCCCTGGGATTTCTTTGGAAGGAATGATGCTGATGCTGAAACTCCAGTACTTTGGCCACCTCATGCAAAGAGTTGACTCATTGGAAAAGACTCTGATGCTGGGAGGGATTGGGGGCAGGAGGAAAATGTGATGACAGAGGATGTGATGTCTGGATGGCATCACTGACTCGATAGACATGAGTCTGTGTGAATTCCGGAGTTGGTGCTGGACAGGGCTGCCTGGTGTGCTGCAATTCATGGGGTTGCAAAGTGTCAGACACAACTGAGCGACTGAACTGAACTGAACTGAACTGGACCTGGCAACAGATCTCCTCTTCTTCCCATCAGTTACTACTAAGTGTCAGACCCCACAATGTGTGGATGACCTAGTCCTGATGGCAGAGACTTGTTCTCAGTGATGGAAAGTGTCAGGAACCACCCAACAATGAGCTGACCTGAGGGAGTGGTGCCCCAGAAGAATAAAGAAAATGATGACTCTGAAATAAAGTGAGGACCACGGGGCTGATGCCATTTACATGCAAAAGCCCAGATCCTAACCCTGCATGCCTTTTATTGTTAACTTCTACTTCCTTATTTGTGCTCTAGAGATAACTGTTTTTATAATCTCAGATGGAGGGTACAGATATACAATGCACAGTCCTGCCTGGTCTACTAAGGGGCAAAGCAAGCAGCCACCCAGGACAATAAAGCAAGCAAACTTATGGTGACACCTGTGGTGTCTAGCCTACTAACTACTCAAGTGACCCCGGTCTTTGACAAGGAAGAAATCACCTGGGCACAAACTGAGTGAGGAACATGGTGGGAGGATGGATGGTGGAAACTCAGATGGGAGACTTTTTGTCCCCTCTAGGTTGGCATTTCAACTCGTTATGAATTTCCATCAATCCATCCATTTAGGTAAAATAAGACTTGCGATAATATCTAACTGTCTTGTGTGCAGATGCAAGCTGCCAATGTATAACCTATTCTAAAAATAACCTAGTTCCAAGGAGACAGCACCTCCTGGAATTCAATTAAAGAGGACAGCTCTATTTAAACATCTACAGGTGGACTTCACTGACATTAAGCCATGCTAAGGATACAAATATTTGCTGGTGATGGTATGTACATTTCCAGAATGGGTGGAAGTTTATCCCACCAAGACTGAAAAAACAAGAAAGTGGCCTGATGTATGCTGAGAGACATTATTTGTAGGTTTGAGTTCCCTTTGAATATAGGATCAGATAATGGGCCTGCATTTATGGTTGAGTTACTTCAACTGGTTTGCAAAACTGTAAATATTAAATGGAAACTACATACAATGTATAGGCCACAAAGCTCAGGAATGGTTCAGAAAATGAACTGGGCTATCAAGGTGACTTTGGAAAAATGAGTGTAAGAAACTGGCACCCCAATCCACCCCCACCCCCATGGATGAACATGCTGTCATTAGCTGCCATTAGTGTTAATGAGGATCAGAATCACACTGCCCCCCTCAACAATCTCATGGGTATTCCCCATATGTGATAATGTTTGGGAGGCCTCCCCCATTTTTCAGAAGTACAGGGAAAATTATCATCAAGAGGAAGAATGGAGGTGTTGTGGCAACTGGAATAGTTGGGGAAGCTGATCCATGATAACCCCTATGTTCAGGAGAGAATTCCATTTTCTCTAGGCACTACTGTACACCTATACTCATCAGGAGATTTAATGCATAAAGAATTGGAAGCAGCAGACATTGTCCCCCATCTGGAAAGGACAACCACAGATCCAGTATGGAGCCACTACTGATGACTCTGCTATTCCTTTTTTTTTTTTTTTAAATGCTTATCTCTCTCTTTTTTTTTTTTTTAACTTTACATAATTGTATTAGTTTTGCCAAATATCAAAATGAATCCGCCACAGGTATACATGTGTTCCCCATCCCGAACCCTCTTCCCTCCTCCCTCCCCATACCATCCCTCTGGGCCATCCTAGTGCACCAGCCCCAAGCATCCAGCATCATGCATCGAACCTGGACTGGCAACTCGTTTCCTACATGATATTTTACATGTTCATGCCATTCTCCCAAATCTTCCCACACTCTCCAGCTCCCACAGAGTCCATAAGACTGTTCTATACATCAGTGTCTCTTTTGCTGTCTCGTACACCAGGTTATTGTTACCCTCTTTCTAAATTCCATATATATGCGTTAGTATACTGTATTTATGTTTTTCCTTCTGGCTTACTTCACTCTGTATAATAGGCTCCAGTTTCATCCACCTCATTAGAACTGATTCAAATGTATTCTTTTTAATGGCTGAGTAATACTCCATTGTGTATATGTACCACTGCTTTCTTATCCATTCATCTGCTGATGGACATCTAGGTTGCTTCCATGTCTTGGCTATTATAAACAGTGCTGCGATGAACATTGGGGTACACGTGTCTCTTTCCCTTCTGGTTTCCTCAGTGTGTATGCCCAGCAGTGGGGTTGCTGGATCATAAGGCAGTTCTATTTCCAGTTTTTTAAGGAATCTCCACACTGTTCTCCATAGTGGCTGTACTAGTTTGCATTCCCACCAACAGTGTAAGAGGGTTCCCTTTTCTCCACACCCTCTCCAGCATTTATTATTTGTAGACTTTTGGATCGCAGCCATTCTGACTGGTGTGAAATGGTACCTCATAGTGGTTTTGATTTGCATTTCTCTGAAAATGAGTGATGTTGAGCATCTTTTCATGTGCTTGTTAGCCATCTGTATGTCTTCTTTGGAGAAATATCTATTTAGTTCTTTGGCCCATTTTTTGATTGGGTCATTTATTTTTCTGGAGTTGAGCTGTAGGAGTTGCTTGTATATTTTTGAGATTAGTTGTTTGTCGGTTGCTTCATTTGCTATTATTTTCTCCCATTCTGAAGGCTGTCTGTTCACCTTGCTAATAGTTTCCTTTGTTCTTCAGAAGCTTTTAAGGTTAATTAGGTCCCATTTGTTTATTTTTGCTTTTATTTCCAATGTTCTGTAGGTGGTTCACTGAGGATCCAAGCTTCACCATGGGAGACGTCACCGGTTCTAGAACCTAGGGAGCTCTGGTACCCACTAGGCGGCCGCCTAGTGAGTCGTATTACGTAGCTTGGCGTAAT

Preferred homologous arms comprise at least about 1 kB of uninterruptedsequence from Table 7, more preferably at least about 2 kB, even morepreferably at least about 4 kB, and even more preferably at least about6 kB. A particularly preferred 6.6 kb bovine sequence (nt 1461 to nt8078 of the 11 kb sequence in Table 7) for use as a homologous arm isprovided below. TABLE 8 6.6 kb bovine homologous arm sequence (SEQ IDNO: 12) GTATACCCGTCTGTGGTAGAACATTTACAAACATTTCTCCTGAATATATGTCTAGGAAAGAAATGAATGGTTTTGTGTATTTTTAGCAAGCTCTTTTTTTTTCTTTTTTTGTAAATTTTGATGAATATTCTTGTATTTTCTAATGAATATCTGCTATATTTTAAAATGTGCCAACTTTTAAAAATATTCATTGGTATGAACTAATACCATGAATTCCAGATGTAATTGGATATGACTCCTTTCTCTACCATTTATCAGGGCTGACATTGATGGATTTGTTTTGGTCCTCAGTTTGTTTACCTTTAATCAGCAGTAGGAATAATAATAAATTAAAATAAAAAACAGCAAAGCAGAAAAATAAATCCTCATTGGGATGCTGGGAAGACTATGTAACTTTAAGGTGTATAATGAATCAATGAGCAAAAATATATAAAGCATTACAATTAAAAGTCAACATTAATTAATGCTAACATTAACTAATTAGTATGTTAACTAACACTAACATACTAACACTAACTAGTATGGATGATAATTATACAAAATAAAAATGACAAAAGCTACCTACTAGGATCTGTGAAAACTAAATAAATGAAGGTCATATTCTGTGAGTGAAAGTATGCATAGTACTCAGAACAGAGATAAGCACTGGTAACTAACAACTGTTGACTGAAAAATGACAAGAGTTGTGAATTAAGTTTTCTTTGGGGCAAAATGAGGACTGCAGCCCAGGAGGCAGCATCAGATAGCTCTAAGAGACTACTCCAAAGTGGCAGTGGGGGAAAGTCAATATATAAGGTTTTGGTGAAGGGGGAGTTCAAAACCATGAACTGCTCATTTTACAAGAGGTTTTTTTGTTAGTCATGAATATCTGATGTCACCATGAGGGGATTTAGTGCTTACTCTATATATGAGGAGATGCAAGTATTGAGATCATACAATGTAATCCTAAAGCATCCATCTATCTAAAGACCTGTCTCGAAGACAGCCTCACCCTGAACTCCCTCAGGGTTGTTGAAGGTCAACAGCATGAGGTTCAATCACCATAGAGGCAGATGGCAAACACCTTTGTTGTTCAGTTGTCGGCCAATGCTCTTGATAGATGCCAATTTGTAGTTGACACAACTAATTAACAGAGAAGGCAATGGCAACATACTCCAGTACCTCTTGCCTGGAAAATCCCATTGGATGGAAGAGCCTGGTAAGCTGCAGTTCTATAGGTCGCAAAGAGTCGGACACCACTAGTGACTTACTCTGACTTTTCAGTTTCAATGCATTGGAGAGAAATGGCACCTACCCCCAATGTTTCTTGGCCTTGGAGAATCCCAGGGATGGCAGAGCCTGGTGGGCTGCTGTCTGTGGGGTCACAAAGAGTCGGACACAACTAAAGCGACTTACCAGCAGCAGCAGCAACAAATTAAAATAATGATAACAAATAACAGTGACAACCTACCTCAATTGGATGCAGAAAGGACAAAATTACTTAAAGATGCATAATAATTCAAGGATTTAAAGTATTTAAAGTACTTAGGGTAGATGTAGGCACTGGTAAAGAAATAAATAGTTAGAATAATTAAAAAAATAAACAGAAGGACAAAAACAAAAACAAAACCTGTTCTGCTTCATTGAGATGCTGTGAAGACTGAAGAAACTATGATGCATACAGATTTAATGAATATTTAATATATTCAGAGGTTAACATTATTAAAGTGCTAAATAGCAATAATGATAATGATGGTAACAATGATAATGATATTATAATAATAAAACCCCTCACTGGAATATTATGAGACTAAATAGGTAAAGGTATGTAAGGTTCAAGAAATAAATATACAATGTTCTTACAGTAAAAGATAACATTAGGAAAGAAGTAATTAATAATAATTACAAATATTAATCATGATAATAAATAACAGCAAATCTTTCCTTCAGGGGATTCTGTGAAGACTAAATATGAAAGTATTTAGATTCAAAGAGTAGATGTATATAATGTACTAAAAATGGAGTTGTTTTATGATGTGTAGCTATAGCAATAATGAAAGCAACAATGACATCATTTGATATGCCTGTGAAGACTGAATAATTTCAAGTGAGCAGAGTTCAAGGAGCACAATGTACTGCAAATTAAGGTCAGTTTTAATAGAGAAAAAATCAATACTAATAATAATTCCAATAGCAATAATAGTACAAATATAGCAATGATGGATACTTAACTAGGATGCTATGAACACTAAGGAAATTAAGACTTAAAGGATTTGATGAGAAAGTGTATCTAAAGTACTAAGAGAAGAAAGTCAACATGAGTAAAATCTAAGTAGTAATAATAATAATTATGAGGATGATGATGATAAAGTAGAAATAAAACCTACTTCAGGGATGCTGTGAAGACTAAGTGAAGGTGTAGGATTCAAGAAATAAGTATTTTGAAATACTTGGAACACCGATAGATATTAGTAAAACACTAATTAATAACACCACCAACATGAATAATAATAAATAATAAAAATGAAACACATCATTGGGATACTATGGCAGTTTTTTAACTAAGTTATGGTATATAGGGGCTGAATGAGTAAATGCATAAAGAAGTACTTAGAAAAGAAGGATTGGAGACAAGATGGCAGACATTTGTCTGAACGTGAAAGAACACTGAATGAACACTGAAAGATGAACTCCCCCAAGTTGGGAGTGACCAATTGCTACTGGAGAAGAGTGGAGAAGAGCTCAGATGAATGAAGAGGCTGAGTCAAAGCAAAACAACTCCAATGGTGTAAAGAAAAATATTTCATAGGAATCTGGAATGTTAGGTCCATGAATCAAGATGTTGGGAAGGCTGGGAGGAGGGAAAGAGAGATTCCATCTTGAAGACTGTCAGTTATCTTAAGGCACGATGAAAACTGGGCCTGAACCCTGTTAACTATTGTCAAACTAAAGTCAGGAAACTCCATCCTCACAGATGGCAAAGATTGGAAGTAAAGGTCAGATTGTGTTAGACTAACGATAGTGCCTGAACGTAAAGGTCAGATTGTGTTAGACTAATGAGAGTGCCTGAACCTGCATGTTGTAGTTGTTAATTCTTCCACACCTGCATATTGTAAAACAAATTACTAATGTGTAACCAGTTTGAGTGAACTCTGGGAGTTGGTGATGGACAGGGAGGCCTGGTGTGCTGTGATTAATGGTGTTGCAAAGAGTCGGACACAACTTAACGACTGAACTGAACTGAACTGAACTGAATGTGTAACCATTCATGTAGTGGAGGGTATAAAACTGAGTCCTCCAAAATCATCAAGGTCCTTGTCAGAACCGATTCCCTTGGGCCTGTTATGTGTAATAAAACTGTTCACTATACTGAGTGTCCTCCAAGGATTGTTCTACAACTCTGGATTCTACAAAATACCTGGTGTGTTGGCTGTGAAATCCTCAGAGAGAGAGGCACATTGAGCCTCCACCTGAGGCTTTCACTGGGATGAAAGCTTCTGTGAGGGGATGGCACCTCCTCTCTTAGATCACCTCTTGTTTTATTGACTCATCTTTCTAAGCAGACTTCACAAGACTGTGGATTACAGAGGGAAACACTCAAGTAGGTCCCACTGTAATAGTGGAAGAAGGGGCCTGATCAACTTATTGGGGCTGGATGAACCTGTGGTGACTGTGTCCTTGTAGGCTCAGTGGGGAATTGTTTACTGAAGTAAGTAAAACACTGTTAACAGAATCTGTGCCAATTGTTTGTCAATGTCTTACTGGTTTCCAAGCGACTGTCCAATTTGTGCAACACCCTCCCATTCTCCTAGGCATTCAGGGACTTCCTGAATGTTGTTTATGACCAGACTGAAACTGAGCTCTGGGTGCACATTTTGTCTGCACTGACCCATAATAAGACAGACTGGGATCTTGCCAGGATCAGACTTATGCCAAAGAGAAGTATATTGAAAGCCTTAATAGATTGGATTTGGATGACTGCTAACAGAAAAACATGAGAGATAATACCATGGAAAGACTTGGTAGGTAAGACCCTTTTCACTTTGATCATAGTTGAGGTACAGTGGCCCTTGTCCTTCTTTGAGTGGAGACTTCAGTCAGGGCTGGGGTACAAGACCCTAGCAATGAGCGATGAAATAGAAGTTGGACTTGCTGTAAGTGATAGAAGGAAAGTAAAAAGTAGGAAAGGTAGCAGAGAATTCAAACCAACCTTTCCTGAATGGATGATTAAAATTTCAAAAGGGTTTTGGAGGAAACAAACAAGCAAAAAGTGAACCCCAGTCAAGCTTAGAACATTCTGTGAGCTAGACTGGCCATTCTTTGGGGTAGGATGGCTCTCAGAAGAACCCTATGATTGGACAAAGTTTGTACTCTGTGGTTACTGAACAGCCAGGACACCCAGACCAGTTCCTGTACTTATGGCTACTAACTGCACAAAATCCACATCGTTAGGCCCAGGTATGTTATCTTGGAAAAGGAGAGAGCAAGATTTTGCTGGTGAAGCAAAATCCAAGAAAGAAAAATTAAAGGAAATATGATCTGGAGGAGTAGGAACCTCCCACCATGCCCTCTCATTCTGGAGAACATGAAGGGGATCCCCAGAAGAAGAGGAAGGGACATTCCTCCTCTGCGTCCCCCAGTGGAGGAGGTTCTCTCTTCCCTCCTATTGTACCAAAAATCGTGACAGGAGCTACTGCATCAACATTATACCCAACCCTCCCCAGTTTAGAAGAGGAGAGAAAGGGAGAGTTAAGTGTTCATAGGCAGCTGAGGTTCTACAAAGGAACCTCAAGAGAGAGAGGTTTTGCAAATACCTTTTACGGAGGTTCAAGCAGTATCACAGGTGGGGCCAGATGGGCATATCCATCCTGGCCACACTGTTCTTTTCTATCAGCCATTCTTTACCACTGGTCTTTTGAACTGGCAAAGGCATACCCCTCCCTATTCTAAGAAACCATATGGTCAATTCATCGGACAAAGATTATTTTCAGGATCACCAACCTGTGTGGGATGACATAGCCCAGCTTCTCCTCACCCTCGTCAGTACAGAAGAAAGACACCGGGTCCTCCCAGAAGTATGTAAATGGCTTCACTGGTAAAATTTCAGGTGGGAATTTTTTCCTCATTTCTGTGGTGATACCAAAGGGAAGATGAGTGGAAGCTCTAACAGTCATCTGGAGGGCTACAGACCCTAGAACCCCTAACAAAGCTGTTTCCTGAAGTATAGGCTAAAAGCAATCCCCTGGACTTGCTGAAAACCACCCTCTGGTGATAATAGAGCTAAAGGTGGGAGCCTAGCCCATAAGGAAAAAACAATATCCTATACCACTGGCTGCCAGGGAGGGAATCAAGCTTTCACATTGATAGGCTGAAGGGCACAGGCATACTGGTGGAATGCCAATCACCATGGAACACCCCTTTCCTCCCAGTAAAGAAGGACAGGGAAAAAGATTATTGGGAAGCTCAGGGCATGAGATCCCAGCGATGGGTCAGAAAACCCAGCTGACTTGGATTCAGGCTACCACAAGGATTCAAAATTTCCTTACAATATTTGGGAGATGCAGACAAAAGAACAAAGCTAGTGGACGTGATGGAATTCCAGTTGAGCTATTTCAAATCCTGAAAGATGATGCTCTGAAAGTGAGGCACTCAATATGCCAGCAAATTTGGAAAACTCAGCAGTGGCCACAGGACTGGAAAAGGTCAGTTTTCATTCCAATCCCAAAGAAAGGCAATGCCAAAGAATGCTCAAACTACCGCACAATTACACTCATCTCACACGCTAGTAAAGTAATGCTCAAAATTCTCCAAGCCAGGCTTCAGCAATACGTGAACTGTGAATTTCCTGATGTTGAAGCTGGTTTTAGAAAAAGCAGAGGAACCAGAGATCAAATTGCCAACATCTGCTGGATCATGGAAAAAGCAAGAGAGTTCTAGAAAAATATTTATTTCTGCTTTATTGTCTATGGAAAAGCCATTGACTGTGTGGATCACAGTACACTGTGGAAAATTCTGAAACAGATGGGAATACCAGACCACTTGACCAGCCTCTTGAGAACTCTGTATGCAGGTCAAGAAGTAACAGTTAGAACTGGACATGGAACAATAGACTGGTTCCAAATAGGAAAAGAAGTACACCAAGGCGGCATATTGTCACCCTGCTTATTTACCGTGCAGAGTACATGCAGAGTACATCATGAGAAATGCTGGACTGGAAGAAACACAAGCTGGAATCAAGATTGCAGGGAGAAATATCAATAACCTCTGATATGCAGATGACACCACCCTTATGGCAGAAAGTGAAGAGGAACTAAAAAGCCGCTTAAAGAAAGTGAAAGTGGAGAGTGAAAAAGTTGGCTTAAAGCTCAACATTCAGAAAACGAAGATCATGGCATCTGGTCCCATCGCTTCATGGGAAAAAGATGGGAAACAGTGTCAGACTTTATTTTGTTGGGCTCCAAAATCACTGCAGATGGTGAGTGCTGCCATGAAATTAAAAGCACTTACTCCCTGGAAGGAAAGTTATGACCAGTTTAGATAGCATATTCAAAACAGAAACATTACTTTGCCAACAAAGGTCCGTCTAGTCAAGGCTATGGTTTTTCCTGTGGTCATATTTGGATGTGAGAGTTG GACTGTGA

In similar fashion, a porcine SRY gene sequence was used to screen aporcine BAC library to isolate a clone containing the porcine SRY. Theprimers used for library screening in the present invention were:PSRYF1: 5′cacctgtgact tagtttcag 3′ (SEQ ID NO: 13) PSRYR1:5′ggctaatcacgggaacaac 3′ (SEQ ID NO: 14)

Sequencing downstream from the 3′ end of the SRY gene on the BAC clone,˜3.8 kb of sequence was obtained. The sequence is shown below in Table9. TABLE 9 Sequence 3′ of the porcine SRY gene (5′ to 3′) (SEQ ID NO:15) ACATGTTTGACCTATAAAGAATTACCGGCATGCCAATATGACTCAACCTGTCTTTACGACTGCTTAAAAGAGCACTACCTTAATAAGAAAGTATCTTAACACACAAACTGCTTGATTTCGAAAACCATCTGTTTTTCCTTCTAATAGAACAATTTTTTTATACCTAATTTTAGTTGTTCCCGTGATTAGCCATTAAGTACGTAACAGTATATATTAGTATTCTGATAATCCTTAGCATAGCTGATAGAATTCTCTTTATTCTCACTGTCAAAACTGTAGTGCTGGGGAGCATGCACAAATTTATGATACAGGAACTTCCATGGAAGTATTTGTACCTAATAAAGCAGTCCCTTGTAGAGTCTGTTCTTTTGTCTTTTCAGCTATTTTGCCTGTCTTTGTAAACTGCAGGTAAAGTAGTGAATATATATGTGTAGTCTATCTGTTTTGAGATTCTTTCTGATATATTGCCTCTCCCAGCTTCAGAAGAGAAAGAAGAGTTTTGCTGCCATTTGCAACTCAGTTCCTTCACTCCGCACAAATCTATGCACTTTGACCTTGAGTTTGAACCATCATGACATCCTTCTGCTAAGACGAAATCTTTTTCTTCTTCTTTTTAATGAAATCTTTAATTGGCTCCTGTGAGACTCACGGTTTCGCGTCTTTTCCGAAAGTTTTCTTTTAACCAGGACCAAATGTTTGTTTCCATTGTCCTCAACTTTGACGTTCTGGGGGGTGCGGGTGGGGAATAGGGATATAATGTTGAGAATATGAACTATTCAGATTGGTGGGGAGGGGCGGGGGGAAGGGGGCATGGGAGGTGGACGAGCCTGTCCGGTTAATCTGGTGAGAAGTCAGTGAAAATGTAAGTCAAAGGCATTATAAAATTTGCCTATGGCCTAAAGTAGAAACTCTGGCAGTTTTCAGAGAAAAGCATCAATTTTTGAAATAAAAATAAGCTGATGGTCTCTTGTCTCTGTATTTATATACCATATGCCAAAATTAACTTTCCAGTGCATATATTCCAAAGCTTTAAAAAAAAAAAAAATTGTCTCAGGTAGTAAAACTCAAAACAGGAAAATGTATGTGGTAGAGTAAAATGTCACGTTTTTGAAAGAAAATACAAGGTAAAACAGGAATTAATTTCACGGACTAATTCGCTCCAGAAACAGTGCTGTTTATTCGGAGATTTACTGCCCCATCTTCCTCTACCCCCGCCCCCGCCCCCGCCCCAGGTTGGGAATTATATGTTGCAATAACCTTTTAAACAACTGTCTAAACTACTCTTAGGTGGAAACTGTGAACAACAAACCTGCCATAAAAGTATCATCATGAGCTATGGGTCTGCTCCGGCCTATACTTGTCACCGCTTTGGTACACTTACCGGACATATTTCTGTCTGTTTAAACTTTGGTCAGCTAAAAATTAAAACTCCCGCCTGGACCAGACCCTAACCACCATCCCATGACTACTGACTAGGAGACTCAACACAGGACCCCTGCCCTATAAAACTTAACTCTCCCTACACAGCAGGAGGGGTGGGGTGGGGAGGGGCTGAGTTCTTTCTCAGTGCTCCTGGCCATCTCTTTGGTCAATAAAATTTGTTTGGGACCTCAGTGTTCCAGTTGACTGTCTTTTCTTCTCTTCTGTGTGTTAACAACTGCTTCGTATTTTTTAATGTCTTTATATACATTTTACACACATATATATGCAAACTGACAGTATTAATGGCCTGAACCTAGCCAGAACTCACATTGGGACTTGAACCCATGATTTTAAATTAGAATCACTCACCCTGTGTCTGGACTCACTGAGGTTCAGGTTCTTCATGTCTCCTCGCAGAAGGAATTCAGCAAGCGACAAAGGGATAGGCAAGAAATAGGTTTATTTTTTTTGGACGCTTGTGAGAGATGCAAGCAGGCAGGCAAGTTCTGCCCCAAGGATCTGAGGATCCAGATAGATGGGTGGGCTACAGTTTTATCCTCCAAGGGGAGTGGAGGTGGGAAAAGCCGGCCTTGGTATCCGGTAAGGTGTGTATTCAAATCAGCAGAAGGGTGGTCCTCAAACTCTTGCCCTTGATCTGAATCTGAATGCAGGCCCCATCCCATCTGCACCCAATGACCTGAGGCAATTCTCACACTTCCACTAGTTAAGCAAGCCTGCCTTGTTCTGATGGCTGTTTTTGAGCAATTTATTTACTTACAGTGGTCTCCCAATATCCCCTAAGTTTTCCTCTTTATCTGTGGTCCTTTACTGGGACCCCTAAAACTTCCTGTGCCTACTCCATCCCTATACATATATATATGTATGTATATATGTATATGTATGTATTTGTGTTTTATGTTTAAAACCTCTCTTTCTGAAACTGACAGCATTTATGACTTGAACCCAGCTAGAGCCCAGACTGGAACTTAACCCACTTTTTTTTCTTTTCTCTTTTTTTTAAATTACGAATCACTCACCCCGGTGTCCAGCCTTACTTGAGGGTCAGGTTCTTTGTGCCTCAGCCAACAGAAAGGAATTCAATGGGAGACAAAGTGATAGTCAAGAAACAGATTTATTAAGACAGGACGCATGAGAGATGTCAAGTGGGCAGGCAAGGAAGCTCTGCCCTGAGGCTTAGGTGGGCTACAGTTTTATCCTCAAGGGGAGTGGAGGTCAGAAAAGCCTGCCTCTTCCTCTTTCTTCCAGTATCTGTTAAGAGAGTGTTTGACCCTGTAAGGTCAAACTAGGACTGTCATGGTGCATGTTCACATCAGCAGAAGGGTGGTCCTTAAACTCCTGCCCTTAGGTCTGAACCTGAATGCAAGCCTCACCACCCCCCCACCCCCTGGCACCCAGTGACCTGAGGCAATTCTCATGGCTCCACCACAGGAGAGCAAGCCTGCCTTATTCTGATGGCTTTCTTGAGCAGTTATTAACTTACAGTGATCTCCCAAAGTTCCCTAGGTTTCCCTCTCTATGGTCTTTTAGGACTTTTACAACTACCCGTGTAACTATCCTACTCCATCCCTATCATCTCCACATGTACAGAAATAACCTCTACTCAGACCTTCATCAAAAAAGATTGATTTTCCTTTTATCAAACTTCACATAAATCACAGCATAAAGTATTATGTCAAGTTTGTTGTGCTTAGTATAATTTCAGTGACATTTCAGCTTGTTTGTCTTAGAAATTACTATGTAATTCCATTCTATTTTTTTATAGACATGTGAATGGACACCTTCTGGTTTTAGCACAAGTACATAGTGTATACATGTCCATGAGAGAACATTTACAGGCATTTCTACTGAGTATATACCTAGGAAAGAAATTGTGTGTTTTTGACACATATTTAGCAGGTATTGATAACAAATATTTTTGTATTTTCTAAAGAATATTTGCCATATCTTCAAATGTCAACATCCAAAAAAATTTAAGGGTATGAACTAAGAGGATGGCCTCCAGAGTCAGTCAGTCTGGACCTGACTCCTTCCCTGCCATTTATTAGGCCAGACACTGAATGATTGCTTCATCTCTCTGGGCCTCAATTTTCTCACTTTTAAGTAAGAAGGAGAAGGAGGAGGAGGAAGAGGAGGAGGGGGGAGGAGGAAGGGGGAGAAGGAGAAAGAGAAGAAGAAGAAGAAGGATAATAATGATAATACTAACGAATGAAAATAAAACAATAAGAACAAAACCAACTTCATTGGGATATTGGGAAGACTAAAAAAGTTAAGGTGTATGATGATTCAATGTACATAAAATATAAAAAGTATATTTAAATAGTATTAAAGAATTTCCTGTCATGGTGCAGTGGCTAGTGAATTGACGTAGGAACCATGAGGTTGTAGGTTCAATCCCTGGCCTCGTTCAG

The skilled artisan will understand that one or more nucleotides may bedeleted, substituted, and/or added to such a sequence while stillproviding a functional homologous arm. Preferred homologous arms arethose in which no more than about 2% of the nucleotides differ bydeletion, substitution, and/or addition from the sequences disclosedherein; more preferably no more than about 1% of the nucleotides differby deletion, substitution, and/or addition from the sequences disclosedbelow; even more preferably no more than about 0.5 % of the nucleotidesdiffer by deletion, substitution, and/or addition from the sequencesdisclosed herein; and most preferably no more than about 0.1% of thenucleotides differ by deletion, substitution, and/or addition from thesequences disclosed herein. The term “about” in this context refers to±10% of a given percentage (e.g., about 1% refers to from 0.9% to 1.1%).

Transfection and Selection of Transgenic Cells

A day 63 bovine male fetus was collected and the genital ridge cellswere obtained by 0.3% protease (Sigma cat. #P6991, St. Loius, Mo.)digestion of the genital ridges for 45 minutes at 37° C. Body cells wereobtained from a partial body (minus head and viscera) trypsin-EDTA (LifeTechnologies cat. #25300-062, Rockville, Md.) digestion for 45 minutesat 37° C. Following digestion and filtration through a 70 μm filter,genital ridge cells were cultured in Amniomax medium (Life Technologiescat. #11269-016) and body cells were cultured in αMEM (Life Technologiescat. #32561-037) supplemented with 10% fetal bovine serum (FBS, Hyclone,Logan, Utah. and 0.1 mM 2-mercaptoethanol.

Prior to transfection by electroporation, cultured genital ridge andbody cells were dissociated using trypsin and counted. The insertionvector is linearized by cutting with Avr II, which cuts the vector inthe Y chromosome arm piece. An aliquot of genital ridge cells (1.2×10⁷)was pelleted by centrifugation, resuspended in 1.0 ml αMEM without serumand divided into two 0.4 cm electroporation cuvettes (BioRadLaboratories, Hercules, Calif.). To each of these cuvettes was added 50μg DNA. The cells were subjected to electroporation using 250V and 960μF (BioRad GenePulser with Capacitance Extender, BioRad Laboratories)and the contents of each cuvette were aliquoted equally into five, 100mm culture dishes and cultured in Amniomax medium. An aliquot of bodycells (1.2×10⁷) was similarly transfected and cultured.

Following 2 days in culture, cells were passaged into selection medium(Amniomax medium containing 600 μg/ml G418, Life Technologies cat.#10131-027). Non-transfected control cells were passaged into selectionmedium at the same time. Following 14 days of selection, the controlcells were dead, while the transfected cells had given rise todrug-resistant colonies. For the genital ridge colonies, the cells weretrypsinized, counted and aliquoted into 96-well plates seeding anaverage of 2 cells per well; drug selection was lowered to 100 μg/ml.The 96-well plates were monitored daily until confluent wells wereobserved. Typically, cells in these wells were passaged into duplicatewells so that cells could be analyzed and if found to be positive,frozen for future nuclear transfer. In populations of bovine body andgenital ridge cells transfected with the mutant BiP-containing vector,PCR analysis indicated that the vector had been incorporated into thegenome of the cells.

Example 2 Cloning Transgenic Porcine Animals

Porcine Oocyte Recovery and Maturation

Sow and gilt ovaries were collected at separate, local abattoirs andmaintained at 30° C. during transport to the laboratory. Folliclesranging from 2-8 mm were aspirated into 50 ml conical centrifuge tubes(BD Biosciences, Franklin Lakes, N.J.) using 18 gauge needles and vacuumset at 100 mm of mercury. Follicular fluid and aspirated oocytes fromsows and gilts were pooled separately and rinsed through EmCon® filters(Iowa Veterinary Supply Company, Iowa Falls, Iowa) with HEPES bufferedTyrodes solution (13iowhittaker, Walkersville, Md.). Oocytes surroundedby a compact cumulus mass were selected and placed into North CarolinaState University (NCSU) 37 oocyte maturation medium (Petters et al., JReprod Fertil Suppl 48, 61-73 (1993)) supplemented with 0.1 mg/mlcysteine (Grupen et al., Biol Reprod 53, 173-178 (1995)), 10 ng/ml EGF(epidermal growth factor) (Grupen et al., Reprod Fertil Dev 9, 571-575(1997)), 10% PFF (porcine follicular fluid) (Naito et al., Gamete Res21, 289-295 (1988)), 0.5 mg/ml cAMP (Funahashi et al., Biol Reprod 57,49-53 (1997)), 10 IU/ml each of PMSG (pregnant mare serum gonadotropin)and hCG (human chorionic gonadotropin) for approximately 22 hours(Funahashi et al., J Reprod Fertil 98, 179-185 (1993)) in humidified airat 38.5° C. and 5% CO₂. Subsequently, they were moved to fresh NCSU 37maturation medium which did not contain cAMP, PMSG or hCG and incubatedfor an additional 22 hours. After approximately 44 hours in maturationmedium, oocytes were stripped of their cumulus cells by vortexing in0.1% hyaluronidase for 1 minute. Sow and gilt derived oocytes were eachused in the in vitro fertilization and nuclear transfer proceduresdescribed below. These procedures were controlled so that comparisonscould be made between sow and gilt derived oocytes for in vitro embryodevelopment, pregnancy initiation rate upon embryo transfer, and littersize upon farrowing.

Nuclear Transfer

Upon removal of cumulus cells, oocytes were placed in CR2 (Rosenkranz etal., Theriogenology 35, 266 (1991)) embryo culture medium that contained1 μg/ml Hoechst 33342 and 7.5 μg/ml cytochalasin B for approximately 30minutes. Micromanipulation of oocytes was performed using glasscapillary microtools in 150 μl drops of TL HEPES on 100 mm dishes (BDBiosciences) covered with light mineral oil. Glass capillary microtoolswere produced using a pipette puller (Sutter Instruments, Novato,Calif.) and microforge (arishige International, East Meadow N.Y.).Metaphase II oocytes were enucleated by removal of the polar body andthe associated metaphase plate. Absence of the metaphase plate wasvisually verified by ultraviolet fluorescence, keeping exposure to aminimum. A single donor cell obtained from a confluent culture bytrrpsin-EDTA dissociation was placed in the perivitelline space of theoocyte so as to contact the oocyte membrane. A single electrical pulseof 95 volts for 45 μsec from an ElectroCell Manipulator 200(Genetronics, San Diego, Calif.) was used to fuse the membranes of thedonor cell and oocyte, forming a cybrid. The fusion chamber consisted ofwire electrodes 500 um apart and the fusion medium was SOR2 (0.25 Msorbitol, 0.1 mM calcium acetate, 0.5 mM magnesium acetate, 0.1% BSA, pH7.2, and osmolarity 250). Following the fusion pulse, cybrids wereincubated in CR2 embryo culture medium for approximately 4 hours priorto activation.

Activation

Oocytes/cybrids were activated by incubation in 15 μM calcium ionomycin(Calbiochem, San Diego, Calif.) for 20 minutes followed by incubationwith 1.9 mM 6-dimethylaminopurine (DMAP) in CR2 for 3-4 hours. AfterDMAP incubation, cybrids were washed through two 35 mm plates containingTL-HEPES, cultured in CR2 medium containing BSA (3 mg/ml) for 48 hours,then placed in NCSU 23 medium containing 0.4% BSA for 24 hours followedby a final culture in NCSU 23 containing 10% FBS. Total time in culturewas for 0-4 days following activation.

Embryo Transfer and Pregnancy Detection

Embryos at various stages of development were surgically transferredinto uteri and/or oviducts of asynchronous recipients essentially asdescribed by Rath (Rath et al., Theriogenology 47, 795-800 (1997)).Briefly, recipients (parity 0 or 1 female porcines) were selected thatexhibited first standing estrus 24 hours after oocyte activation to 24hours prior to oocyte activation. For surgical embryo transfer,recipients were anesthetized with a combination of 2 mg/kg ketamine,0.25 mg/kg tiletamine/zolazepam, 1 mg/kg xylazine and 0.03 mg/kgatropine (Iowa Veterinary Supply). Anesthesia was maintained with 3%halothane (Iowa Veterinary Supply). While in dorsal recumbence, therecipients were aseptically prepared for surgery and a caudal ventralincision was made to expose and examine the reproductive tract. Embryosthat were cultured less than 48 hours (1-2 cell stage) were placed inthe ampullar region of the oviduct by feeding a 5.5-inch TomCat®catheter (Sherwood Medical) through the ovarian fimbria. Embryoscultured 48 hours or more (≧4 cell stage) were placed in the tip of theuterine horn using a similar catheter. Typically, 100-300 NT embryoswere placed in the oviduct or uterine tip, depending on embryonic stageand 100 IVF embryos were placed in the oviduct. All recipients andprotocols conformed to University of Wisconsin animal health-careguidelines. Ultrasound detection of pregnancy was accomplished using anAloka 500 ultrasound scanner (Aloka Co. Ltd, Wallingford, Conn.) with anattached 3.5 MHz trans-abdominal probe. Monitoring for pregnancyinitiation began at 23 days post fusion/fertilization and repeated asnecessary through day 40. Pregnant recipients were reexamined byultrasound weekly.

Example 3 Cloning Transgenic Bovine Animals

Embryo Construction

Oocytes aspirated from ovaries were matured overnight (about 16-18hours) in maturation medium. Medium 199 (Biowhittaker, Cat #12-119F)supplemented with luteinizing hormone 10IU/ml (LH; Sigma, Cat #L9773), 1mg/ml estradiol (Sigma, Cat #E8875) and 10% FCS or estrus cow serum, wasused.

Oocytes were stripped of their cumulus cell layers and nuclear materialstained with Hoechst 33342 5mg/ml (Sigma, Cat #2261) in TL HEPESsolution supplemented with cytochalasin B (7 μg/ml, Sigma, Cat #C6762)for 15 min. Oocytes were then enucleated in TL HEPES solution undermineral oil. A single nuclear donor cell of optimal size (12 to 15 μm)was then inserted from a cell suspension and injected into theperivitelline space of the enucleated oocyte. The cell and oocytemembranes were then induced to fuse by electrofusion in a 500 μm chamberby application of an electrical pulse of 90V for 15 μs, forming acybrid.

3-4 hours following cybrid formation, cybrid activation was induced by a4 min exposure to 5 μM calcium ionophore A23187 (Sigma Cat. #C-7522) orionomycin Ca-salt in HECM (hamster embryo culture medium) containing 1mg/ml BSA followed by a 1:1000 dilution in HECM containing 30 mg/ml BSAfor 5 min. For HECM medium, see, e.g., Seshagiri & Barister, 1989,“Phosphate is required for inhibition of glucose of development ofhamster eight-cell embryos in vitro,” Biol. Reprod. 40: 599-606. Thisstep was followed by incubation in CR2 medium containing 1.9 mM6-dimethylaminopurine (DMAP; Sigma product, Cat #D2629) for 4 hrsfollowed by a wash in HECM and then culture in CR2 media with BSA (3mg/ml) under humidified air with 5% CO₂ at 39° C. For CR2 medium, see,e.g., Rosenkrans & First, 1994, “Effect of free amino acids and vitaminson cleavage and developmental rate of bovine zygotes in vitro,” J. Anim.Sci. 72: 434-437. Mitotic divisions of the cybrid formed an embryo.Three days later the embryos were transferred to CR2 media containing10% FCS for the remainder of their in vitro culture.

Second Nuclear Transfer (Recloning)

Embryos from the first generation NT at the morula stage weredisaggregated either by pronase E (1-3 mg/ml in TL HBEPES) ormechanically after treatment with cytochalasin B. Single blastomereswere placed into the perivitelline space of enucleated aged oocytes(28-48 hours of incubation). Aged oocytes were produced by incubatingmatured “young” oocytes for an additional time in CR2 media with 3 mg/mlBSA in humidified air with 5% CO₂ at 39° C.

A blastomere from a nuclear transfer embryo was fused into theenucleated oocyte via electrofusion in a 500 μm chamber with anelectrical pulse of 105V for 15 μs in an isotonic sorbitol solution(0.25 M) at 30° C. Aged oocytes were simultaneously activated with afusion pulse, not by chemical activation as with young oocytes.

After blastomere-oocyte fusion, the cybrids from the first or secondgeneration NT were cultured in CR2 media supplemented with BSA (3 mg/ml)under humidified air with 5% CO₂ at 39° C. On the third day of culture,developing embryos were evaluated and cultured further until day sevenin CR2 media containing 10% FCS. Morphologically good to fair qualityembryos were non-surgically transferred into recipient females.

Example 4 In Vitro Fertilization

Matured oocytes were inseminated by the procedures described by Long etal. (Theriogenology 51, 1375-1390 (1999)) with a modification describedby Grupen and Nottle (Theriogenology 53, 422 (2000)). Briefly, 50matured oocytes stripped of their cumulus and in a volume of 3 μl, wereplaced into 92 μl drops of fertilization medium (TLP-PVA). Each dropcontaining oocytes was inseminated with 5 μl of fertilization mediumcontaining 2000 sperm. Fresh boar semen was purchased from GenesDiffusion (Stoughton, Wis.). Several different boars were used duringthe course of these experiments. After 10 minutes of co-incubation withsperm, the oocytes were moved to a fresh drop of fertilization mediumand incubated for an additional 5 hours. Oocytes were washed throughunused fertilization drops to remove sperm and cultured in NCSU 23 with0.4% BSA until embryos were transferred into recipients 0-4 dayspost-fertilization. Embryos that were maintained in culture to evaluatedevelopment rates were placed in NCSU 23 with 10% FBS from day 5 to day7.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The cell lines, embryos,animals, and processes and methods for producing them are representativeof preferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Modifications therein andother uses will occur to those skilled in the art. These modificationsare encompassed within the spirit of the invention and are defined bythe scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described. The nucleotide sequences describedherein are provided without corresponding homologous sequences accordingto the Watson/Crick base pairing rules. Those of skill in the art willrecognize that the corresponding homologous sequences are also describedherein.

Other embodiments are set forth within the following claims.

1. A mammal comprising a transgene on a sex chromosome, wherein theexpression of said transgene is operably linked to a promoter regionthat confers haploid-specific expression to said transgene.
 2. Themammal of claim 1, wherein said promoter also confers tissue-specificexpression to said transgene.
 3. The mammal of claim 2, wherein saidtissue-specific expression is testis-specific expression.
 4. The mammalof claim 3, wherein said transgene is expressed in one or more cellsselected from the group consisting of primary spermatocytes, secondaryspermatocytes, spermatids, and spermatozoa.
 5. The mammal of claim 1wherein said promoter region comprises the promoter for the protaminegene.
 6. The mammal of claim 1 wherein expression of said transgeneselectively kills those cells expressing said transgene.
 7. The mammalof claim 1 wherein expression of said transgene selectively disablesthose cells expressing said transgene.
 8. The mammal of claim 1 whereinsaid transgene encodes a marker protein which can be used to sort thosecells expressing said transgene from cells not expressing saidtransgene.
 9. Haploid cells harvested from the mammal of claim
 1. 10.Haploid cells according to claim 9 which have been enriched for cellsexpressing said transgene.
 11. The mammal of claim 1 wherein the mammalis an ungulate.
 12. The mammal of claim 1 wherein the mammal is selectedfrom the group consisting of porcine, ovine, bovine, and caprine. 13.The mammal of claim 1, wherein expression of said transgene isinducible.
 14. The mammal of claim 13, wherein expression of saidtransgene selectively kills those cells expressing said transgene whenexposed to an inducing agent.
 15. The mammal of claim 13, whereinexpression of said transgene selectively disables those cells expressingsaid transgene when exposed to an inducing agent.
 16. The mammal ofclaim 13, wherein said transgene encodes a marker protein which can beused to sort those cells expressing said transgene when exposed to aninducing agent from cells not expressing said transgene.
 17. Haploidcells harvested from the mammal of claim
 13. 18. Haploid cells harvestedfrom the mammal of claim 13 which have been enriched for cellsexpressing said transgene.
 19. A method for producing a population ofmammalian haploid cells that is enriched for haploid cells containing aspecific sex chromosome, wherein said method comprises: harvestinghaploid cells from a mammal comprising a transgene which is capable ofkilling or disabling cells in cis when expressed, wherein said transgeneis selectively expressed in cells comprising said specific sexchromosome, and wherein the expression of said transgene is operablylinked to a promoter region that confers haploid-specific expression tosaid transgene, whereby expression of the transgene kills or disablesthose haploid cells expressing said transgene.
 20. The method of claim19, wherein said method further comprises removing or discarding saidkilled or disabled haploid cells.
 21. A method for producing apopulation of mammalian haploid cells that is enriched for haploid cellscontaining a specific sex chromosome, wherein said method comprises: (a)harvesting haploid cells from a mammal comprising a transgene which iscapable of killing or disabling cells in cis when expressed, whereinsaid transgene is selectively expressed in cells comprising saidspecific sex chromosome, wherein the expression of said transgene isoperably linked to a promoter region that confers haploid-specificexpression to said transgene, and wherein expression of said transgeneis inducible; and (b) inducing the expression of said transgene to killor disable the those haploid cells expressing said transgene.
 22. Themethod of claim 19, wherein said method further comprises removing ordiscarding said killed or disabled haploid cells.
 23. A method forproducing a population of mammalian haploid cells that is enriched forhaploid cells containing a specific sex chromosome, wherein said methodcomprises (a) harvesting haploid cells from a mammal comprising atransgene which is capable of generating a detectable phenotype in cellsin cis when expressed, wherein said transgene is selectively expressedin cells comprising said specific sex chromosome, and wherein theexpression of said transgene is operably linked to a promoter regionthat confers haploid-specific expression to said transgene, wherebyexpression of the transgene produces said detectable phenotype marker inthose haploid cells expressing said transgene; and (b) sorting thehaploid cells based on the expression of said detectable phenotype. 24.A method for producing a population of mammalian haploid cells that isenriched for haploid cells containing a specific sex chromosome, whereinsaid method comprises (a) harvesting haploid cells from an animalcomprising a transgene which is capable of generating a detectablephenotype in cells in cis when expressed, wherein said transgene isselectively expressed in cells comprising said specific sex chromosome,wherein the expression of said transgene is operably linked to apromoter region that confers haploid-specific expression to saidtransgene, and wherein expression of said transgene is inducible,whereby expression of the transgene produces said detectable phenotypein those haploid cells expressing said transgene; and (b) sorting thehaploid cells based on the expression of said detectable phenotype. 25.The method of any one of claims 19-24, wherein the mammal is anungulate.
 26. The method of claim 25 wherein the ungulate is selectedfrom the group consisting of porcine, ovine, bovine, and caprine. 27.The method of any one of claims 19-24, wherein the haploid cellsharvested are spermatozoa.
 28. A method for producing a mammal,comprising contacting an ovum with one or more spermatozoa producedaccording to the method of claim 27 to fertilize said ovum.
 29. A methodaccording to claim 28, wherein said ovum is fertilized by an assistedreproductive technique.